Resting-state EEG features modulated by depressive state in healthy individuals: insights from theta PSD, theta-beta ratio, frontal-parietal PLV, and sLORETA.

Depressive states in both healthy individuals and those with major depressive disorder exhibit differences primarily in symptom severity rather than symptom type, suggesting that there is a spectrum of depressive symptoms. The increasing prevalence of mild depression carries lifelong implications, emphasizing its clinical and social significance, which parallels that of moderate depression. Early intervention and psychotherapy have shown effective outcomes in subthreshold depression. Electroencephalography serves as a non-invasive, powerful tool in depression research, with many studies employing it to discover biomarkers and explore underlying mechanisms for the identification and diagnosis of depression. However, the efficacy of these biomarkers in distinguishing various depressive states in healthy individuals and in understanding the associated mechanisms remains uncertain. In our study, we examined the power spectrum density and the region-based phase-locking value in healthy individuals with various depressive states during their resting state. We found significant differences in neural activity, even among healthy individuals. Participants were categorized into high, middle, and low depressive state groups based on their response to a questionnaire, and eyes-open resting-state electroencephalography was conducted. We observed significant differences among the different depressive state groups in theta- and beta-band power, as well as correlations in the theta-beta ratio in the frontal lobe and phase-locking connections in the frontal, parietal, and temporal lobes. Standardized low-resolution electromagnetic tomography analysis for source localization comparing the differences in resting-state networks among the three depressive state groups showed significant differences in the frontal and temporal lobes. We anticipate that our study will contribute to the development of effective biomarkers for the early detection and prevention of depression.

Synthetic DNA nanopores for direct molecular transmission between lipid vesicles.

Lipid vesicles hold potential as artificial cells in bottom-up synthetic biology, and as tools in drug delivery and biosensing. Transmitting molecular signals is a key function for vesicle-based systems. One strategy to achieve this function is by releasing molecular signals from vesicles through nanopores. Nevertheless, in this strategy, an excess of molecular signals may be required to reach the targets, due to the dispersion of the signals during diffusion. The key to achieving the efficient utilization of signals is to shorten the distance between the sender vesicle and the target. Here, we present a pair of DNA nanopores that can connect and form a direct molecular pathway between vesicles. The nanopores are self-assembled from nine single DNA strands, including six 14-nucleotide single-stranded overhangs as sticky-end segments, enabling them to bind with each other. Incorporating nanopores shortens the distance between different populations of vesicles, allowing less diffusion of molecules into bulk solution. To further reduce the loss of molecules, a DNA nanocap is added to one of the nanopore's openings. The nanocap can be removed through the toehold-mediated DNA strand displacement when the nanopore meets its counterpart. Our DNA nanopores provide a novel molecular transmission tool to lipid vesicles-based systems.

Depressive states in healthy subjects lead to biased processing in frontal-parietal ERPs during emotional stimuli.

Subthreshold depressive (sD) states and major depression are considered to occur on a continuum, and there are only quantitative and not qualitative differences between depressive states in healthy individuals and patients with depression. sD is showing a progressively increasing prevalence and has a lifelong impact, and the social and clinical impacts of sD are no less than those of major depressive disorder (MDD). Because depression leads to biased cognition, patients with depression and healthy individuals show different visual processing properties. However, it remains unclear whether there are significant differences in visual information recognition among healthy individuals with various depressive states. In this study, we investigated the event-related potentials (ERPs) and event-related spectrum perturbation (ERSP) of healthy individuals with various depressive states during the perception of emotional visual stimulation. We show that different neural activities can be detected even among healthy individuals. We divided healthy participants into high, middle, and low depressive state groups and found that participants in a high depressive state had a lower P300 amplitude and significant differences in fast and slow neural responses in the frontal and parietal lobes. We anticipate our study to provide useful parameters for assessing the evaluation of depressive states in healthy individuals.

Dimethyl Sulfoxide-Free Cryopreservation of Differentiated Human Neuronal Cells.

In recent years, cells provided by cell banks and medical facilities have been used for cell therapy, regenerative therapy, and fundamental research. Cryopreservation is an effective means of maintaining stable cell quality over a long period of time. The slow freezing method is most suitable for processing many human cells isolated simultaneously from organs and tissues, but it is necessary to develop a freezing solution for this method. In this study, we report the successful development of a dimethyl sulfoxide (DMSO)-free freezing medium for differentiated neuronal cells. Neuronal differentiation results in the differentiation of undifferentiated SK-N-SH cells into neuronal cells. A basic freezing medium (BFM) was prepared using Dulbecco's modified Eagle's medium, 1 M maltose, and 1% sericin as the essential ingredients, supplemented with 5%-40% propylene glycol (PG). Each BFM supplemented with 5%-40% PG was evaluated in undifferentiated cells. After thawing, BFM supplemented with 10% and 20% PG were 83% and 88% viable, respectively. There was no significant difference between the 10% and 20% PG groups. However, a significant difference was observed when the concentration of PG in the BFM decreased by 5% (5% PG vs. 10% PG; p = 0.0026). Each DMSO-free BFM was evaluated using differentiated neuronal cells. There was no significant difference between the 10% PG BFM and stem-CB-free groups. Viability was significantly different in the 10% glycerol BFM (4.8%) and 10% PG BFM (45%) (p = 0.028). The differentiated cells with 10% PG BFM showed higher adherence to culture dishes than those with 10% glycerol BFM. These results show that BFM containing PG was effective in differentiating neuronal cells. DMSO affects the central nervous system at low concentrations. This report indicates that DMSO is unsuitable for neuronal cells with multipotent differentiation potential. Therefore, it is essential for cell banking and transplantation medicine services to select appropriate cell freezing media.

Functional Evaluation of 3D Liver Models Labeled with Polysaccharide Functionalized Magnetic Nanoparticles.

Establishing a rapid in vitro evaluation system for drug screening is essential for the development of new drugs. To reproduce tissues/organs with functions closer to living organisms, in vitro three-dimensional (3D) culture evaluation using microfabrication technology has been reported in recent years. Culture on patterned substrates with controlled hydrophilic and hydrophobic regions (Cell-ableTM) can create 3D liver models (miniature livers) with liver-specific Disse luminal structures and functions. MRI contrast agents are widely used as safe and minimally invasive diagnostic methods. We focused on anionic polysaccharide magnetic iron oxide nanoparticles (Resovist®) and synthesized the four types of nanoparticle derivatives with different properties. Cationic nanoparticles (TMADM) can be used to label target cells in a short time and have been successfully visualized in vivo. In this study, we examined the morphology of various nanoparticles. The morphology of various nanoparticles showed relatively smooth-edged spherical shapes. As 3D liver models, we prepared primary hepatocyte-endothelial cell heterospheroids. The toxicity, CYP3A, and albumin secretory capacity were evaluated in the heterospheroids labeled with various nanoparticles. As the culture period progressed, the heterospheroids labeled with anionic and cationic nanoparticles showed lower liver function than non-labeled heterospheroids. In the future, there is a need to improve the method of creation of artificial 3D liver or to design a low-invasive MRI contrast agent to label the artificial 3D liver.

Cryopreservation of undifferentiated and differentiated human neuronal cells.

The effective use of human-derived cells that are difficult to freeze, such as parenchymal cells and differentiated cells from stem cells, is crucial. A stable supply of damage-sensitive cells, such as differentiated neuronal cells, neurons, and glial cells can contribute considerably to cell therapy. We developed a serum-free freezing solution that is effective for the cryopreservation of differentiated neuronal cells. The quality of the differentiated and undifferentiated SK-N-SH cells was determined based on cell viability, live-cell recovery rate, and morphology of cultured cells, to assess the efficacy of the freezing solutions. The viability and recovery rate of the differentiated SK-N-SH neuronal cells were reduced by approximately 1.5-folds compared to that of the undifferentiated SK-N-SH cells. The viability and recovery rate of the differentiated SK-N-SH cells were remarkably different between the freezing solutions containing 10% DMSO and that containing 10% glycerol. Cryoprotectants such as fetal bovine serum (FBS), antifreeze proteins (sericin), and sugars (maltose), are essential for protecting against freeze damage in differentiated neuronal cells and parenchymal cells. Serum-free alternatives (sericin and maltose) could increase safety during cell transplantation and regenerative medicine. Considering these, we propose an effective freezing solution for the cryopreservation of neuronal cells.

A Study of the Effects of Plasma Surface Treatment on Lipid Bilayers Self-Spreading on a Polydimethylsiloxane Substrate under Different Treatment Times.

Plasma-treated poly(dimethylsiloxane) (PDMS)-supported lipid bilayers are used as functional tools for studying cell membrane properties and as platforms for biotechnology applications. Self-spreading is a versatile method for forming lipid bilayers. However, few studies have focused on the effect of plasma treatment on self-spreading lipid bilayer formation. In this paper, we performed lipid bilayer self-spreading on a PDMS surface with different treatment times. Surface characterization of PDMS treated with different treatment times is evaluated by AFM and SEM, and the effects of plasma treatment of the PDMS surface on lipid bilayer self-spreading behavior is investigated by confocal microscopy. The front-edge velocity of lipid bilayers increases with the plasma treatment time. By theoretical analyses with the extended-DLVO modeling, we find that the most likely cause of the velocity change is the hydration repulsion energy between the PDMS surface and lipid bilayers. Moreover, the growth behavior of membrane lobes on the underlying self-spreading lipid bilayer was affected by topography changes in the PDMS surface resulting from plasma treatment. Our findings suggest that the growth of self-spreading lipid bilayers can be controlled by changing the plasma treatment time.

Preparation of Size-controlled Giant Vesicles Under Physiological Conditions.

Giant vesicles (GVs) are model cell membranes that function as tools for the study of cell membrane properties. Recently, researchers have been calling for GVs of specific sizes for use in studies with precise needs. In this paper, we report a method of forming GVs of specific sizes by using an agarose-swelling approach. The resulting GVs had a narrow size distribution and were successfully formed under physiological conditions.

Self-spreading method for forming lipid bilayer on a patterned agarose gel: Toward precise lipid bilayer patterning.

Forming artificial cell membranes is a suitable strategy for studying drug responses of membrane proteins. In order to form lipid bilayer with both mechanical stability and membrane protein functions, hydrogel supported bilayer has attracted attentions. Combinational use of self-extraction method for lipid bilayer formation and agarose gel patterning should realize hydrogel-supported bilayer with any shape and large area. In this study, we aimed to form a lipid bilayer on a patterned agarose gel and to characterize the membrane. First, lipid mixture was attached on an agarose gel, and lipid layers spread on the gel surface. With fluorescent observation, it is suggested that thin lipid layer was formed on the agarose gel, and their distance-dependent changes in spreading velocity was consistent with that in lipid bilayer. Next, the lipid layer was characterized with fluorescence recovery after photo breaching experiment. As a result, it is indicated that lipid molecules in the lipid layer on the agarose showed lateral diffusion, a typical characteristic of lipid bilayer. Taken together, we confirmed that lipid bilayer can be formed on the patterned agarose gel with self-spreading method. The hydrogel-supported bilayer will be a suitable tool for drug discovery.

Enhanced Adipogenic Differentiation of Human Adipose-Derived Stem Cells in an In Vitro Microenvironment: The Preparation of Adipose-Like Microtissues Using a Three-Dimensional Culture.

The application of stem cells for cell therapy has been extensively studied in recent years. Among the various types of stem cells, human adipose tissue-derived stem cells (ASCs) can be obtained in large quantities with relatively few passages, and they possess a stable quality. ASCs can differentiate into a number of cell types, such as adipose cells and ectodermal cells. We therefore focused on the in vitro microenvironment required for such differentiation and attempted to induce the differentiation of human stem cells into microtissues using a microelectromechanical system. We first evaluated the adipogenic differentiation of human ASC spheroids in a three-dimensional (3D) culture. We then created the in vitro microenvironment using a 3D combinatorial TASCL device and attempted to induce the adipogenic differentiation of human ASCs. The differentiation of human ASC spheroids cultured in maintenance medium and those cultured in adipocyte differentiation medium was evaluated via Oil red O staining using lipid droplets based on the quantity of accumulated triglycerides. The differentiation was confirmed in both media, but the human ASCs in the 3D cultures contained higher amounts of triglycerides than those in the 2D cultures. In the short culture period, greater adipogenic differentiation was observed in the 3D cultures than in the 2D cultures. The 3D culture using the TASCL device with adipogenic differentiation medium promoted greater differentiation of human ASCs into adipogenic lineages than either a 2D culture or a culture using a maintenance medium. In summary, the TASCL device created a hospitable in vitro microenvironment and may therefore be a useful tool for the induction of differentiation in 3D culture. The resultant human ASC spheroids were "adipose-like microtissues" that formed spherical aggregation perfectly and are expected to be applicable in regenerative medicine as well as cell transplantation.

Direct Gaze Estimation Based on Nonlinearity of EOG.

Electrooculography (EOG) is one of the measures used to estimate the direction of a person's gaze; however, conventional EOG techniques suffer from a drift issue which makes it difficult to extract an accurate absolute eye angle. The technique proposed here is based on the nonlinearity of the EOG and offers a practical solution to this problem. It estimates the absolute eye angles before and after a saccade, which cancels the offset due to the drift. Additionally, it does not require any effort from the user or any target, but instead uses only the difference of the EOGs. Experiments with five subjects confirm that the proposed technique can estimate the absolute eye angle with an error of less than 4(°). They also show improvements are achieved with several options such as weighting and multiple saccades. The technique will contribute to practical EOG-based interaction systems.

Orientation-modulated attention effect on visual evoked potential: Application for PIN system using brain-computer interface.

This research demonstrates the orientation-modulated attention effect on visual evoked potential. We combined this finding with our previous findings about the motion-modulated attention effect and used the result to develop novel visual stimuli for a personal identification number (PIN) application based on a brain-computer interface (BCI) framework. An electroencephalography amplifier with a single electrode channel was sufficient for our application. A computationally inexpensive algorithm and small datasets were used in processing. Seven healthy volunteers participated in experiments to measure offline performance. Mean accuracy was 83.3% at 13.9 bits/min. Encouraged by these results, we plan to continue developing the BCI-based personal identification application toward real-time systems.

Three-Dimensional In Vitro Hepatic Constructs Formed Using Combinatorial Tapered Stencil for Cluster Culture (TASCL) Device.

Attempts to create artificial liver tissue from various cells have been reported as an alternative method for liver transplantation and pharmaceutical testing. In the construction of artificial liver tissue, the selection of the cell source is the most important factor. However, if an appropriate environment (in vitro/in vivo) cannot be provided for various cells, it is not possible to obtain artificial liver tissue with the desired function. Therefore, we focused on the in vitro environment and produced liver tissues using MEMS technology. In the present study, we report a combinatorial TASCL device to prepare 3D cell constructs in vitro. The TASCL device was fabricated with an overall size of 10 mm × 10 mm with microwells and a top aperture (400 µm × 400 µm, 600 µm × 600 µm, 800 µm × 800 µm) and bottom aperture (40 µm × 40 µm, 80 µm × 80 µm, 160 µm × 160 µm) per microwell. The TASCL device can be easily installed on various culture dishes with tweezers. Using plastic dishes as the bottom surface of the combinatorial TASCL device, 3D hepatocyte constructs of uniform sizes (about ɸ 100 µm-ɸ 200 µm) were produced by increasing the seeding cell density of primary mouse hepatocytes. The 3D hepatocyte constructs obtained using the TASCL device were alive and secreted albumin. On the other hand, partially adhered primary mouse hepatocytes exhibited a cobblestone morphology on the collagen-coated bottom of the individual microwells using the combinatorial TASCL device. By changing the bottom substrate of the TASCL device, the culture environment of the cell constructs was easily changed to a 3D environment. The combinatorial TASCL device described in this report can be used quickly and simply. This device will be useful for preparing hepatocyte constructs for application in drug screening and cell medicine.

Spheroid Formation and Evaluation of Hepatic Cells in a Three-Dimensional Culture Device.

In drug discovery, it is very important to evaluate liver cells within an organism. Compared to 2D culture methods, the development of 3D culture techniques for liver cells has been successful in maintaining long-term liver functionality with the formation of a hepatic-specific structure. The key to performing drug testing is the establishment of a stable in vitro evaluation system. In this article, we report a Tapered Stencil for Cluster Culture (TASCL) device developed to create liver spheroids in vitro. The TASCL device will be applied as a toxicity evaluation system for drug discovery. The TASCL device was created with an overall size of 10 mm × 10 mm, containing 400 microwells with a top aperture (500 µm × 500 µm) and a bottom aperture (300 µm diameter circular) per microwell. We evaluated the formation, recovery, and size of HepG2 spheroids in the TASCL device. The formation and recovery were both nearly 100%, and the size of the HepG2 spheroids increased with an increase in the initial cell seeding density. There were no significant differences in the sizes of the spheroids among the microwells. In addition, the HepG2 spheroids obtained using the TASCL device were alive and produced albumin. The morphology of the HepG2 spheroids was investigated using FE-SEM. The spheroids in the microwells exhibited perfectly spherical aggregation. In this report, by adjusting the size of the microwells of the TASCL device, uniform HepG2 spheroids were created, and the device facilitated more precise measurements of the liver function per HepG2 spheroid. Our TASCL device will be useful for application as a toxicity evaluation system for drug testing.

Investigation of P300 response characteristics through human color vision-based visual stimulation.

In this study, we propose visual stimulation based on the primary colors (red, green, and blue) in order to investigate the characteristics of the P300 response. Eleven healthy volunteers participated in our experiment, and their brain signals were recorded by electroencephalography (EEG). Using two basic measures referred to as `on-peak' and `off-peak' for comparison of the P300 response among the participants, we found that the P300 response varies depending on the color of the stimulus. The results of this investigation are expected to contribute to various existing and future EEG-based applications.

Diagnosis method of mild cognitive impairment based on power variance of EEG.

Mild cognitive impairment (MCI) patients and healthy people were classified by using a "power variance function (PVF)", namely, an index of electroencephalography (EEG) proposed in a previous report. PVF is defined by calculating variance of the power variability of an EEG signal at each frequency of the signal using wavelet transform. After confirming that the distribution of PVFs of the subjects was a normal distribution at each frequency, the distributions of PVFs of 25 MCI patients and those of 57 healthy people were compared in terms of Z-score. The comparison results indicate that for the MCI patients, the PVFs in the θ band are significantly higher in left parieto-occipital area and that those in the ß band are lower in the bitemporal area. Multidimensional discriminant analysis using the PVF in the θ-ß band recorded only on four electrodes on the left parieto-occipital area could be used to classify MCI patients from healthy people with leave-one-out accuracy of 87.5%. This indicates the possibility of diagnosing MCI by using EEG signals recorded only on a few electrodes.

Automatic drift calibration for EOG-based gaze input interface.

A drift calibration technique for DC-coupled EOG (electrooculogram) systems is proposed. It assumes a non-linear relationship between EOG and eye angle and estimates the absolute eye angle by the EOG differences during saccade. Drift is calibrated every saccade without user's explicit action, so it is especially suitable for long-term gaze input interfaces. An experiment confirms that it can estimate horizontal absolute eye angle with an error of about 5° in addition to accurate eye movement.

Relating plastic changes of short latency human soleus stretch reflex to changes in task performance induced by training.

Recent findings in the field of neurophysiology showed that operant conditioning on the human H-Reflex is possible. This leads to many possible clinical applications as well as possible sophisticated training methods for athletes. Although stretch reflexes have been subject to extensive literature, knowledge about the influence of short latency stretch reflexes on task performance is lacking. Within this study an ankle control task was designed where perturbations in the magnitude of functional relevance were applied. Results analyzing angle over time after perturbation confirm previous findings which used to analyze the EMG and force response to ankle perturbations. Further it was found that after training the response to perturbations shifted from initially containing latencies which indicate conscious support by transcortical pathways to latencies which could only origin from unconscious stretch reflex responses. The trend of the short latency response to shift towards the long latency response and to diminish, while pre-defined performance criteria improved, denote a functional relevance of the short latency stretch reflex to task performance. Whereas short latency reflexes have any importance at all or if improvements emerge only out of enhancements in the long latency response future work making use of operant conditioning on the short latency H-Reflex will have to unravel.

Research of the characteristics of Alzheimer's disease using EEG.

In this paper, we propose a new method for diagnosing Alzheimer's disease (AD) on the basis of electroencephalograms (EEG). The method, which is termed "Power Variance Function (PVF) method", indicates the variance of the power at each frequency. By using the proposed method, the power of EEG at each frequency was calculated using Wavelet transform, and the corresponding variances were defined as PVF. After the PVF histogram of 42 healthy people was approximated as a Generalized Extreme Value (GEV) distribution, we evaluated the PVF of 10 patients with AD and 10 patients with mild cognitive impairment (MCI). As a result, the values for all AD and MCI subjects were abnormal. In particular, the PVF in the theta band for MCI patients was abnormally high, and the PVF in the alpha band for AD patients was low.