Biological neurons act as generalization filters in reservoir computing.

Reservoir computing is a machine learning paradigm that transforms the transient dynamics of high-dimensional nonlinear systems for processing time-series data. Although the paradigm was initially proposed to model information processing in the mammalian cortex, it remains unclear how the nonrandom network architecture, such as the modular architecture, in the cortex integrates with the biophysics of living neurons to characterize the function of biological neuronal networks (BNNs). Here, we used optogenetics and calcium imaging to record the multicellular responses of cultured BNNs and employed the reservoir computing framework to decode their computational capabilities. Micropatterned substrates were used to embed the modular architecture in the BNNs. We first show that the dynamics of modular BNNs in response to static inputs can be classified with a linear decoder and that the modularity of the BNNs positively correlates with the classification accuracy. We then used a timer task to verify that BNNs possess a short-term memory of several 100 ms and finally show that this property can be exploited for spoken digit classification. Interestingly, BNN-based reservoirs allow categorical learning, wherein a network trained on one dataset can be used to classify separate datasets of the same category. Such classification was not possible when the inputs were directly decoded by a linear decoder, suggesting that BNNs act as a generalization filter to improve reservoir computing performance. Our findings pave the way toward a mechanistic understanding of information representation within BNNs and build future expectations toward the realization of physical reservoir computing systems based on BNNs.

Enhanced responses to inflammatory cytokine interleukin-6 in micropatterned networks of cultured cortical neurons.

Cortical neurons in dissociated cultures are an indispensable model system for pharmacological research that provides insights into chemical responses in well-defined environments. However, cortical neurons plated on homogeneous substrates develop an unstructured network that exhibits excessively synchronized activity, which occasionally masks the consequences induced by external substances. Here, we show that hyperactivity and excessive synchrony in cultured cortical networks can be effectively suppressed by growing neurons in microfluidic devices. These devices feature a hierarchically modular design that resembles the in vivo network. We focused on interleukin-6, a pro-inflammatory cytokine, and assessed its acute and chronic effects. Fluorescence calcium imaging of spontaneous neural activity for up to 20 days of culture showed detectable modulation of collective activity events and neural correlation in micropatterned neurons, which was not apparent in neurons cultured on homogeneous substrates. Our results indicate that engineered neuronal networks provide a unique platform for detecting and understanding the fundamental effects of biochemical compounds on neuronal networks.

Model-free idealization: Adaptive integrated approach for idealization of ion-channel currents.

Single-channel electrophysiological recordings provide insights into transmembrane ion permeation and channel gating mechanisms. The first step in the analysis of the recorded currents involves an "idealization" process, in which noisy raw data are classified into two discrete levels corresponding to the open and closed states of channels. This provides valuable information on the gating kinetics of ion channels. However, the idealization step is often challenging in cases of currents with poor signal-to-noise ratios and baseline drifts, especially when the gating model of the target channel is not identified. We report herein on a highly robust model-free idealization method for achieving this goal. The algorithm, called adaptive integrated approach for idealization of ion-channel currents (AI2), is composed of Kalman filter and Gaussian mixture model clustering and functions without user input. AI2 automatically determines the noise reduction setting based on the degree of separation between the open and closed levels. We validated the method on pseudo-channel-current datasets that contain either computed or experimentally recorded noise. We also investigated the relationship between the noise reduction parameter of the Kalman filter and the cutoff frequency of the low-pass filter. The AI2 algorithm was then tested on actual experimental data for biological channels including gramicidin A, a voltage-gated sodium channel, and other unidentified channels. We compared the idealization results with those obtained by the conventional methods, including the 50%-threshold-crossing method.

Lateral voltage as a new input for artificial lipid bilayer systems.

In this work, we propose lateral voltage as a new input for use in artificial lipid bilayer systems in addition to the commonly used transmembrane voltage. To apply a lateral voltage to bilayer lipid membranes, we fabricated electrode-equipped silicon and Teflon chips. The Si chips could be used for photodetector devices based on fullerene-doped lipid bilayers, and the Teflon chips were used in a study of the ion channel functions in the lipid bilayer. The findings indicate that the lateral voltage effectively regulates the transmembrane current, in both ion-channel-incorporated and fullerene-incorporated lipid bilayer systems, suggesting that the lateral voltage is a practicable and useful additional input for use in lipid bilayer systems.

Filtration-processed biomass nanofiber electrodes for flexible bioelectronics.

An increasing demand for bioelectronics that interface with living systems has driven the development of materials to resolve mismatches between electronic devices and biological tissues. So far, a variety of different polymers have been used as substrates for bioelectronics. Especially, biopolymers have been investigated as next-generation materials for bioelectronics because they possess interesting characteristics such as high biocompatibility, biodegradability, and sustainability. However, their range of applications has been restricted due to the limited compatibility of classical fabrication methods with such biopolymers. Here, we introduce a fabrication process for thin and large-area films of chitosan nanofibers (CSNFs) integrated with conductive materials. To this end, we pattern carbon nanotubes (CNTs), silver nanowires, and poly (3,4-ethylenedioxythiophene):poly (styrenesulfonate) (PEDOT:PSS) by a facile filtration process that uses polyimide masks fabricated via laser ablation. This method yields feedlines of conductive material on nanofiber paper and demonstrates compatibility with conjugated and high-aspect-ratio materials. Furthermore, we fabricate a CNT neural interface electrode by taking advantage of this fabrication process and demonstrate peripheral nerve stimulation to the rapid extensor nerve of a live locust. The presented method might pave the way for future bioelectronic devices based on biopolymer nanofibers.

Bactericidal Activity of Bulk Nanobubbles through Active Oxygen Species Generation.

We investigated the bactericidal activity of bulk nanobubbles (NBs) using E. coli, a model bacterium. Bulk NBs were produced by forcing gas through a porous alumina membrane with an ordered arrangement of nanoscale straight holes in contact with water. NBs with different gas contents, including CO2, O2, and N2, were generated and evaluated for their bactericidal effects. The survival rate of E. coli was significantly reduced in a suspension of CO2-containing NB (CO2-NB water). The N2-NB water demonstrated a small amount of bactericidal behavior, but its impact was not as significant as that of CO2-NB water. When E. coli was retained in O2-NB water, the survival rate was even higher than that in pure water (PW). We investigated the generation of reactive oxygen species (ROS) in NB suspensions by electron spin resonance spectroscopy. The main ROS generated in the NB water were hydroxyl radicals and OH·, and the production of ROS was the strongest in CO2-NB water, which was consistent with the results of the bactericidal effect measurements. We assumed that NB mediated by ROS would exhibit bactericidal behavior and proposed a kinetic model to explain the retention time variation of the survival rate. The results calculated based on the proposed model matched closely with the experimental results.

Optical transport of sub-micron lipid vesicles along a nanofiber.

Enhanced manipulation and analysis of bio-particles using light confined in nano-scale dielectric structures has proceeded apace in the last several years. Small mode volumes, along with the lack of a need for bulky optical elements give advantages in sensitivity and scalability relative to conventional optical manipulation. However, manipulation of lipid vesicles (liposomes) remains difficult, particularly in the sub-micron diameter regime. Here we demonstrate the optical trapping and transport of sub-micron diameter liposomes along an optical nanofiber using the nanofiber mode's evanescent field. We find that nanofiber diameters below a nominal diffraction limit give optimal results. Our results pave the way for integrated optical transport and analysis of liposome-like bio-particles, as well as their coupling to nano-optical resonators.

Bactericidal Activity of TiO2 Nanotube Thin Films on Si by Photocatalytic Generation of Active Oxygen Species.

The photocatalytic bactericidal activity of titanium dioxide (TiO2) thin films has been extensively studied. In this study, we investigated the bactericidal activities of TiO2 nanotube (NT) thin films using Escherichia coli and Staphylococcus aureus cells as the model bacteria. Metallic titanium (Ti) thin films were anodized on a silicon (Si) wafer substrate to form TiO2 NT thin films. To evaluate the bactericidal activity of the TiO2 NT thin films, bacteria on the TiO2 NT thin films were irradiated with near-ultraviolet light (UV-A) at a wavelength of 365 nm. The bactericidal activity was estimated by the survival rate derived from the number of live cells, which form colonies on the cell culture medium. We demonstrated that the survival rate of the two types of bacteria investigated in this study was significantly reduced by UV light irradiation and that there was a difference in the temporal change in the survival rate between the two types of bacteria. Furthermore, we investigated the generation of reactive oxygen species (ROSs) by UV light irradiation of TiO2 NT thin films using electron spin resonance spectroscopy and fluorescence analysis. We found that the main ROS generated on the surface of the TiO2 NT film was the hydroxyl radical, OH•. In addition, the generation of ROSs increased with an increase in the UV irradiation time. We proposed a kinetic model that reproduces the dependence of bacterial viability on the UV light irradiation time by considering the temporal change in the amount of ROSs generated by UV light irradiation. A comparison of the calculated and experimental results revealed that the bactericidal effect consisted of the direct photolysis of bacteria and the photocatalysis via the generation of hydroxyl radicals, with the latter exhibiting a stronger bactericidal effect than the former.

Advances in Artificial Cell Membrane Systems as a Platform for Reconstituting Ion Channels.

An artificial cell membrane that is composed of bilayer lipid membranes (BLMs) with transmembrane proteins incorporated within them represents a well-defined system for the analysis of membrane proteins, especially ion channel proteins that are major targets for drug design. Because the BLM system has a high compatibility with recently developed cell-free expression systems, it has attracted attention as a next-generation drug screening system. However, three issues associated with BLM systems, i. e., their instability, the need for non-volatile organic solvents and a low efficiency of ion channel incorporation, have limited their use as a drug screening platform. In this personal account, we discuss our recent approaches to address these issues based on microfabrication. We also discuss the potential for using the BLM system combined with cell-free expression systems as a drug screening system for future personalized medicine.

Suppression of hypersynchronous network activity in cultured cortical neurons using an ultrasoft silicone scaffold.

The spontaneous activity pattern of cortical neurons in dissociated culture is characterized by burst firing that is highly synchronized among a wide population of cells. The degree of synchrony, however, is excessively higher than that in cortical tissues. Here, we employed polydimethylsiloxane (PDMS) elastomers to establish a novel system for culturing neurons on a scaffold with an elastic modulus resembling brain tissue, and investigated the effect of the scaffold's elasticity on network activity patterns in cultured rat cortical neurons. Using whole-cell patch clamp to assess the scaffold effect on the development of synaptic connections, we found that the amplitude of excitatory postsynaptic current, as well as the frequency of spontaneous transmissions, was reduced in neuronal networks grown on an ultrasoft PDMS with an elastic modulus of 0.5 kPa. Furthermore, the ultrasoft scaffold was found to suppress neural correlations in the spontaneous activity of the cultured neuronal network. The dose of GsMTx-4, an antagonist of stretch-activated cation channels (SACs), required to reduce the generation of the events below 1.0 event per min on PDMS substrates was lower than that for neurons on a glass substrate. This suggests that the difference in the baseline level of SAC activation is a molecular mechanism underlying the alteration in neuronal network activity depending on scaffold stiffness. Our results demonstrate the potential application of PDMS with biomimetic elasticity as cell-culture scaffold for bridging the in vivo-in vitro gap in neuronal systems.

Establishment of a cell-free translation system from rice callus extracts.

Eukaryotic in vitro translation systems require large numbers of protein and RNA components and thereby rely on the use of cell extracts. Here we established a new in vitro translation system based on rice callus extract (RCE). We confirmed that RCE maintains its initial activity even after five freeze-thaw cycles and that the optimum temperature for translation is around 20°C. We demonstrated that the RCE system allows the synthesis of hERG, a large membrane protein, in the presence of liposomes. We also showed that the introduction of a bicistronic mRNA based on 2A peptide to RCE allowed the production of two distinct proteins from a single mRNA. Our new method thus facilitates laboratory-scale production of cell extracts, making it a useful tool for the in vitro synthesis of proteins for biochemical studies.

Mean-field analysis of directed modular networks.

We considered a modular network with a binomial degree distribution and related the analytical relationships of the network properties (modularity, average clustering coefficient, and small-worldness) with structural parameters that define the network, i.e., number of nodes, number of modules, average node degree, and ratio of intra-modular to total connections. Even though modular networks are universally found in real-world systems and are consequently of broad interest in complex network science, the relationship between network properties and structural parameters has not yet been formulated. Here, we show that a series of equations for predicting the network properties can be related using a mean-field connectivity matrix that is defined on the basis of the structural parameters in the network generation algorithm. The theoretical results are then compared with values calculated numerically using the original connectivity matrix and are found to agree well, except when the connections between modules are sparse. Representation of the structure of the network using simple parameters is expected to be conducive for elucidating the structure-dynamics relationship.

Amphiphobic Septa Enhance the Mechanical Stability of Free-Standing Bilayer Lipid Membranes.

Artificial bilayer lipid membranes (BLMs) provide well-defined systems for investigating the fundamental properties of membrane proteins, including ion channels, and for screening the effect of drugs that act on them. However, the application of this technique is limited due to the low stability and low reconstitution efficiency of the process. We previously reported on improving the stability of BLM based on the fabrication of microapertures having a tapered edge in SiO2/Si3N4 septa and efficient ion channel incorporation based on vesicle fusion accelerated by a centrifugal force. Although the BLM stability and incorporation probability were dramatically improved when these approaches were used, some BLMs were ruptured when subjected to a centrifugal force. To further improve the BLM stability, we investigated the effect of modifying the surface of the SiO2/Si3N4 septa on the stability of BLM suspended in the septa. The modified surfaces were characterized in terms of hydrophobicity, lipophobicity, and surface roughness. Diffusion coefficients of the lipid monolayers formed on the modified surfaces were also determined. Highly fluidic lipid monolayers were formed on the amphiphobic substrates that had been modified with long-chain perfluorocarbons. Free-standing BLMs formed in amphiphobic septa showed a much higher mechanical stability, including tolerance to water movement and applied centrifugal forces with and without proteoliposomes, than those formed in the septa that had been modified with a short alkyl chain. These results demonstrate that highly stable BLMs are formed when the surface of the septa has amphiphobic properties. Because highly fluidic lipid monolayers that are formed on the septa seamlessly connect with BLMs in a free-standing region, the high fluidity of the lipids contributes to decreasing potential damage to BLMs when mechanical stresses are applied. This approach to improve the BLM stability increases the experimental efficiency of the BLM systems and will contribute to the development of high-throughput platforms for functional assays of ion channel proteins.

Astrocytes with previous chronic exposure to amyloid ß-peptide fragment 1-40 suppress excitatory synaptic transmission.

Synaptic dysfunction and neuronal death are responsible for cognitive and behavioral deficits in Alzheimer's disease (AD). It is well known that such neurological abnormalities are preceded by long-term exposure of amyloid ß-peptide (Aß) and/or hyperphosphorylated tau prior. In addition to the neurological deficit, astrocytes as a major glial cell type in the brain, significantly participate in the neuropathogenic mechanisms underlying synaptic modulation. Although astrocytes play a significant key role in modulating synaptic transmission, little is known on whether astrocyte dysfunction caused by such long-term Aß exposure affects synapse formation and function. Here, we show that synapse formation and synaptic transmission are attenuated in hippocampal-naïve neurons co-cultured with astrocytes that have previously experienced chronic Aß1-40 exposure. In this abnormal astrocytic condition, hippocampal neurons exhibit decrements of evoked excitatory post-synaptic currents (EPSCs) and miniature EPSC frequency. Furthermore, size of readily releasable synaptic pools and number of excitatory synapses were also significantly decreased. Contrary to these negative effects, release probability at individual synapses was significantly increased in the same astrocytic condition. Taken together, our data indicate that lower synaptic transmission caused by astrocytes previously, and chronically, exposed to Aß1-40 is attributable to a small number of synapses with higher release probability.

Reconstitution of Human Ion Channels into Solvent-free Lipid Bilayers Enhanced by Centrifugal Forces.

Artificially formed bilayer lipid membranes (BLMs) provide well-defined systems for functional analyses of various membrane proteins, including ion channels. However, difficulties associated with the integration of membrane proteins into BLMs limit the experimental efficiency and usefulness of such BLM reconstitution systems. Here, we report on the use of centrifugation to more efficiently reconstitute human ion channels in solvent-free BLMs. The method improves the probability of membrane fusion. Membrane vesicles containing the human ether-a-go-go-related gene (hERG) channel, the human cardiac sodium channel (Nav1.5), and the human GABAA receptor (GABAAR) channel were formed, and the functional reconstitution of the channels into BLMs via vesicle fusion was investigated. Ion channel currents were recorded in 67% of the BLMs that were centrifuged with membrane vesicles under appropriate centrifugal conditions (14-55 × g). The characteristic channel properties were retained for hERG, Nav1.5, and GABAAR channels after centrifugal incorporation into the BLMs. A comparison of the centrifugal force with reported values for the fusion force revealed that a centrifugal enhancement in vesicle fusion was attained, not by accelerating the fusion process but by accelerating the delivery of membrane vesicles to the surface of the BLMs, which led to an increase in the number of membrane vesicles that were available for fusion. Our method for enhancing the probability of vesicle fusion promises to dramatically increase the experimental efficiency of BLM reconstitution systems, leading to the realization of a BLM-based, high-throughput platform for functional assays of various membrane proteins.

Radical generation and bactericidal activity of nanobubbles produced by ultrasonic irradiation of carbonated water.

Our previous study showed that nanobubbles (NBs) encapsulating CO2 gas have bactericidal activity due to reactive oxygen species (ROS) (Yamaguchi et al., 2020). Here, we report that bulk NBs encapsulating CO2 can be efficiently generated by ultrasonically irradiating carbonated water using a piezoelectric transducer with a frequency of 1.7 MHz. The generated NBs were less than 100 nm in size and had a lifetime of 500 h. Furthermore, generation of ROS in the NB suspension was investigated using electron spin resonance spectroscopy and fluorescence spectrometry. The main ROS was found to be the hydroxyl radical, which is consistent with our previous observations. The bactericidal activity lasted for at least one week. Furthermore, a mist generated by atomizing the NB suspension with ultrasonic waves was confirmed to have the same bactericidal activity as the suspension itself. We believe that the strong, persistent bactericidal activity and radical generation phenomenon are unique to NBs produced by ultrasonic irradiation of carbonated water. We propose that entrapped CO2 molecules strongly interact with water at the NB interface to weaken the interface, and high-pressure CO2 gas erupts from this weakened interface to generate ROS with bactericidal activity.

Reconstitution of human ether-a-go-go-related gene channels in microfabricated silicon chips.

This paper reports on the reconstitution of human ether-a-go-go-related gene (hERG) channels in artificial bilayer lipid membranes (BLMs) formed in micropores fabricated in silicon chips. The hERG channels were isolated from Chinese hamster ovary cell lines expressing the channels and incorporated into the BLMs formed by a process in which the two lipid monolayers were folded into the micropores. The characteristic features of hERG channels reported by the patch-clamp method, including single-channel conductance, voltage dependence, sensitivity to typical drugs and dependence on the potassium concentration, were investigated in the BLM reconstitution system. The BLM with hERG channels incorporated exhibited a lifetime of ~65 h and a tolerance to repetitive solution exchanges. Such stable BLMs containing biological channels have the potential for use in a variety of applications, including high-throughput drug screening for various ion-channel proteins.

Two-dimensional water-molecule-cluster layers at nanobubble interfaces.

HYPOTHESIS: Bulk nanobubbles (NBs) have high surface charge densities and long lifetimes. Despite several attempts to understand the lifetime of NBs, their interfacial layer structure remains unknown. It is hypothesized that a specific interfacial layer exists with a hydrogen bond network that stabilizes NBs. EXPERIMENTS: In situ infrared reflectance-absorption spectroscopy and density functional theory were used to determine the interfacial layer structure of NBs. Furthermore, nuclear magnetic resonance spectroscopy was used to examine the interfacial layer hardness of bubbles filled with N2, O2, and CO2, which was expected to depend on the encapsulated gas species. FINDINGS: The interfacial layer was composed of three-, four-, and five-membered ring clusters of water molecules. An interface model was proposed in which a two-dimensional layer of clusters with large electric dipole moments is oriented toward the endohedral gas, and the hydrophobic surface is adjacent to the free water. The interfacial layer hardness was dependent on the interaction with the gas (N2 > O2 > CO2), which supports the proposed interface model. These findings can be generalized to the structure of water at gas-water interfaces.