An Instant Donning Multi-Channel EEG Headset (with Comb-Shaped Dry Electrodes) and BCI Applications.

We developed a new type of electroencephalogram (EEG) headset system with comb-shaped electrodes that enables the wearer to quickly don and utilize it in daily life. Two models that can measure EEG signals using up to eight channels have been implemented. The electrodes implemented in the headsets are similar to a comb and are placed quickly by wiping the hair (as done with a comb) using the headset. To verify this headset system, donning time was measured and three brain computer interface (BCI) application experiments were conducted. Alpha rhythm-based, steady-state visual evoked potential (SSVEP)-based, and auditory steady state response (ASSR)-based BCI systems were adopted for the validation experiments. Four subjects participated and ten trials were repeated in the donning experiment. The results of the validation experiments show that reliable EEG signal measurement is possible immediately after donning the headsets without any preparation. It took approximately 10 s for healthy subjects to don the headsets, including an earclip with reference and ground electrodes. The results of alpha rhythm-based BCI showed 100% accuracy. Furthermore, the results of SSVEP-based and ASSR-based BCI experiments indicate that performance is sufficient for BCI applications; 95.7% and 76.0% accuracies were obtained, respectively. The results of BCI paradigm experiments indicate that the new headset type is feasible for various BCI applications.

Physiological Signal Monitoring Bed for Infants Based on Load-Cell Sensors.

Ballistocardiographs (BCGs), which record the mechanical activity of the heart, have been a subject of interest for several years because of their advantages in providing unobtrusive physiological measurements. BCGs could also be useful for monitoring the biological signals of infants without the need for physical confinement. In this study, we describe a physiological signal monitoring bed based on load cells and assess an algorithm to extract the heart rate and breathing rate from the measured load-cell signals. Four infants participated in a total of 13 experiments. As a reference signal, electrocardiogram and respiration signals were simultaneously measured using a commercial device. The proposed automatic algorithm then selected the optimal sensor from which to estimate the heartbeat and respiration information. The results from the load-cell sensor signals were compared with those of the reference signals, and the heartbeat and respiration information were found to have average performance errors of 2.55% and 2.66%, respectively. The experimental results verify the positive feasibility of BCG-based measurements in infants.

Cell-engineered virus-mimetic nanovesicles for vaccination against enveloped viruses.

Enveloped viruses pose a significant threat to human health, as evidenced by the recent COVID-19 pandemic. Although current vaccine strategies have proven effective in preventing viral infections, the development of innovative vaccine technologies is crucial to fortify our defences against future pandemics. In this study, we introduce a novel platform called cell-engineered virus-mimetic nanovesicles (VNVs) and demonstrate their potential as a vaccine for targeting enveloped viruses. VNVs are generated by extruding plasma membrane-derived blebs through nanoscale membrane filters. These VNVs closely resemble enveloped viruses and extracellular vesicles (EVs) in size and morphology, being densely packed with plasma membrane contents and devoid of materials from other membranous organelles. Due to these properties, VNVs express viral membrane antigens more extensively and homogeneously than EVs expressing the same antigen. In this study, we produced severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) VNVs expressing the SARS-CoV-2 Spike glycoprotein (S) on their surfaces and assessed their preclinical efficacy as a COVID-19 vaccine in experimental animals. The administration of VNVs successfully stimulated the production of S-specific antibodies both systemically and locally, and immune cells isolated from vaccinated mice displayed cytokine responses to S stimulation.

Enhanced decision-making in nicotine dependent individuals who abstain: A computational analysis using Hierarchical Drift Diffusion Modeling.

BACKGROUND: Variability in decision-making capacity and reward responsiveness may underlie differences in the ability to abstain from smoking. Computational modeling of choice behavior, as with the Hierarchical Drift Diffusion Model (HDDM), can help dissociate reward responsiveness from underlying components of decision-making. Here we used the HDDM to identify which decision-making or reward-related parameters, extracted from data acquired in a reward processing task, contributed to the ability of people who smoke that are not seeking treatment to abstain from cigarettes during a laboratory task. METHODS: 80 adults who smoke cigarettes completed the Probabilistic Reward Task (PRT) - a signal detection task with a differential reinforcement schedule - following smoking as usual, and the Relapse Analogue Task (RAT) - a task in which participants could earn money for delaying smoking up to 50min - after a period of overnight abstinence. Two cohorts were defined by the RAT; those who waited either 0-min (n=36) or the full 50-min (n=44) before smoking. RESULTS: PRT signal detection metrics indicated all subjects learned the task contingencies, with no differences in response bias or discriminability between the two groups. However, HDDM analyses indicated faster drift rates in 50-min vs. 0-min waiters. CONCLUSIONS: Relative to those who did not abstain, computational modeling indicated that people who abstained from smoking for 50min showed faster evidence accumulation during reward-based decision-making. These results highlight the importance of decision-making mechanisms to smoking abstinence, and suggest that focusing on the evidence accumulation process may yield new targets for treatment.

Multifluorescence Single Extracellular Vesicle Analysis by Time-Sequential Illumination and Tracking.

We demonstrate a fluorescence-based nanoparticle tracking analysis (NTA) system for the characterization of both the size and membrane protein expression of individual extracellular vesicles (EVs). A sheet of lasers with four different wavelengths was sequentially shone onto extracellular vesicles according to a preprogrammed schedule, providing scattering images intercalated by three fluorescent images. The presence of extracellular vesicles was tracked frame by frame from scattering images. Fluorescence-labeled membrane proteins on EVs were detected by comparing scattering and fluorescent images. The tetraspanins (CD9, CD63, and CD81) of individual HEK293 EVs analyzed by both NTA and total internal reflection fluorescence microscopy showed that the proposed NTA system can contribute to the understanding of individual extracellular vesicles.

Single-vesicle imaging and co-localization analysis for tetraspanin profiling of individual extracellular vesicles.

Extracellular vesicles (EVs) are secreted nano-sized vesicles that contain cellular proteins, lipids, and nucleic acids. Although EVs are expected to be biologically diverse, current analyses cannot adequately characterize this diversity because most are ensemble methods that inevitably average out information from diverse EVs. Here we describe a single vesicle analysis, which directly visualizes marker expressions of individual EVs using a total internal-reflection microscopy and analyzes their co-localization to investigate EV subpopulations. The single-vesicle imaging and co-localization analysis successfully illustrated the diversity of EVs and revealed distinct patterns of tetraspanin expressions. Application of the analysis demonstrated similarities and dissimilarities between the EV fractions that had been acquired from different conventional EV isolation methods. The analysis method developed in this study will provide a new and reliable tool for investigating characteristics of single EVs, and the findings of the analysis might increase understanding of the characteristics of EVs.

Heterogeneous Subcellular Origin of Exosome-Mimetic Nanovesicles Engineered from Cells.

Cell-engineered nanovesicles (CNVs) are considered as an alternative to exosomes, because they can be produced efficiently on a large scale and have been successfully reported in several applied research studies. However, CNVs may originate from various organelles, i.e., some of them may cause adverse effects on recipient cells, and their origin has not yet been identified. In this study, we air-sprayed human embryonic kidney 293 (HEK293) cells into lipid-bilayer CNVs. To identify the subcellular origin of the CNVs, we prepared nine different HEK293 cell lines by transfection with organelle-specific fluorescent protein plasmids that target the plasma membrane, peroxisome, lysosome, early endosome, late endosome, nucleus, mitochondrion, Golgi apparatus, and endoplasmic reticulum. The origin of CNVs were identified by measuring fluorescence expressions for organelle-specific markers using fluorescence nanoparticle tracking analysis (NTA). In the results, we found that CNVs derived from the plasma membrane constituted the largest portion, but CNVs derived from the other organelles comprised a non-negligible portion as well. This information will be useful to guide advanced research on outer membrane vesicles and exosome-mimetic nanovesicles engineered from cells.

Brainstem BOLD response to visual and acoustic stimuli.

Understanding the fundamental roles of brainstem function resulting in proper motor control is critical to motor-rehabilitation after brain injuries. In particular, vestibular and reticular formation nuclei are thought to be associated with spasticity in chronic stroke patients. We used two kinds of stimuli in 10 healthy subjects to activate these nuclei while collecting high-resolution (1.5-mm) fMRI across the majority of brainstem. Optokinetic stimuli evoked illusory self-motion to activate the vestibular nuclei. Acoustic-startle stimuli were sets of loud tones designed to activate of the reticular formation. We summarized the response represented in a form of activation volume, mean percent signal change, and the phase delay (time lag) following the stimulus. We observed patterns of significant activations in the brainstem but did not find significant differences between the stimulus. We conclude that more sensitive measurement techniques are needed to reliably detect vestibular and reticular formation nuclei responses.

Mesenchymal Stem Cell Engineered Nanovesicles for Accelerated Skin Wound Closure.

We report development and characterization of cell-engineered nanovesicles made from mesenchymal stem cells (MSCNVs), which have more than 300 times higher productivity than natural extracellular vesicles (EVs). MSCNVs had similar morphological characteristics to MSCEVs but have molecular characteristics that more resemble MSCs than MSCEVs. In vitro MSCNV treatment increased the proliferation and migration of primary skin fibroblasts and showed better effects than treatment using natural MSCEVs. Quantitative real-time PCR analysis showed increased expression of growth factors in MSCNV-treated skin fibroblasts. Intraperitoneal injection of MSCNVs into syngeneic mice induced mild local inflammation, which resulted in recruitment of immune cells to the injection site. In vivo MSCNV treatment of a mouse skin wound accelerated its healing; this acceleration by MSCNVs may occur by promoting blood vessel formation at the wound site. These results indicate the promise of MSCNVs as agents for regenerative medicine.

Analysis of Extracellular Vesicles Using Coffee Ring.

Extracellular vesicles are categorized in subsets according to their biogenesis processes. To facilitate the investigation of subsets, an effective method is needed for isolating subpopulations. The efficacy of existing density and size-based isolation methods is limited, and as a result, the correlation of properties within separated subpopulations is modest. Here, we introduced size separation with ∼48 nm resolution that exploits Marangoni flow and the coffee-ring effect in microdroplets in which extracellular vesicles are spatially deposited at different location according to size of extracellular vesicle. Interestingly, the analysis of tetraspanin proteins of the extracellular vesicles facilitated by this method reveals that the size of extracellular vesicles is correlated with expression of tetraspanin proteins (CD9, CD63, CD81) that are associated with the size of extracellular vesicles. The findings show that CD9 and CD81 are uniformly expressed regardless of size, CD63 is highly expressed only in larger extracellular vesicles. This evidence indicates that extracellular vesicles can be classified based on size and expression of CD63.

Effect of Concentrated Fibroblast-Conditioned Media on In Vitro Maintenance of Rat Primary Hepatocyte.

The effects of concentrated fibroblast-conditioned media were tested to determine whether hepatocyte function can be maintained without direct contact between hepatocytes and fibroblasts. Primary rat hepatocytes cultured with a concentrated conditioned media of NIH-3T3 J2 cell line (final concentration of 55 mg/ml) showed significantly improved survival and functions (albumin and urea) compared to those of control groups. They also showed higher expression levels of mRNA, albumin and tyrosine aminotransferase compared to hepatocyte monoculture. The results suggest that culture with concentrated fibroblast-conditioned media could be an easy method for in vitro maintenance of primary hepatocytes. They also could be contribute to understand and analyze co-culture condition of hepatocyte with stroma cells.

Formation and manipulation of cell spheroids using a density adjusted PEG/DEX aqueous two phase system.

Various spheroid formation techniques have been widely developed for efficient and reliable 3-D cell culture research. Although those efforts improved many aspects of spheroid generation, the procedures became complex and also required unusual laboratory equipment. Many recent techniques still involve laborious pipetting steps for spheroid manipulation such as collection, distribution and reseeding. In this report, we used a density-controlled polyethylene glycol and dextran aqueous two phase system to generate spheroids that are both consistent in size and precisely size-controllable. Moreover, by adding a few drops of fresh medium to the wells the contain spheroids, they can be simply settled and attached to the culture surface due to reduced densities of the phases. This unique attribute of the technique significantly reduces the numerous pipetting steps of spheroid manipulation to a single pipetting; therefore, the errors from those steps are eliminated and the reliability and efficiency of a research can be maximized.

High-yield isolation of extracellular vesicles using aqueous two-phase system.

Extracellular vesicles (EVs) such as exosomes and microvesicles released from cells are potential biomarkers for blood-based diagnostic applications. To exploit EVs as diagnostic biomarkers, an effective pre-analytical process is necessary. However, recent studies performed with blood-borne EVs have been hindered by the lack of effective purification strategies. In this study, an efficient EV isolation method was developed by using polyethylene glycol/dextran aqueous two phase system (ATPS). This method provides high EV recovery efficiency (~70%) in a short time (~15 min). Consequently, it can significantly increase the diagnostic applicability of EVs.

Microfluidic fabrication of cell-derived nanovesicles as endogenous RNA carriers.

Exosomes/microvesicles are known to shuttle biological signals between cells, possibly by transferring biological signal components such as encapsulated RNAs and proteins, plasma membrane proteins, or both. Therefore exosomes are being considered for use as RNA and protein delivery vehicles for various therapeutic applications. However, living cells in nature secrete only a small number of exosomes, and procedures to collect them are complex; these complications impede their use in mass delivery of components to targeted cells. We propose a novel and efficient method that forces cells through hydrophilic microchannels to generate artificial nanovesicles. These mimetic nanovesicles contain mRNAs, intracellular proteins and plasma membrane proteins, and are shaped like cell-secreted exosomes. When recipient cells are exposed to nanovesicles from embryonic stem cells, mRNAs of Oct 3/4 and Nanog are transferred from embryonic stem cells to the target cells. This result suggests that mimetic nanovesicles can be used as vehicles to deliver RNA. This nanovesicle formation method is expected to be used in exosome research and to have applications in drug and RNA-delivery systems.