Ken Shimono

A new planar multielectrode array for extracellular recording: application to hippocampal acute slice.

The present paper describes a new planar multielectrode array (the MED probe) and its electronics (the MED system) which perform electrophysiological studies on acute hippocampal slices. The MED probe has 64 planar microelectrodes, is covered with a non-toxic, uniform insulation layer, and is further coated with polyethylenimine and serum. The MED probe is shown to be appropriate for both stimulation and recording. In particular, multi-channel recordings of field EPSPs obtained by stimulating with a pair of planar microelectrodes were established for rat hippocampal acute slices. The recordings were stable for 6 h. Finally a spatial distribution of long-term potentiation was studied using the MED system.

Urinary Volatile Compounds as Biomarkers for Lung Cancer

Lung cancer is a leading cause of deaths in cancer. Hence, developing early-stage diagnostic tests that are non-invasive, highly sensitive, and specific is crucial. In this study, we investigated to determine whether biomarkers derived from urinary volatile organic compounds (VOCs) can be used to discriminate between lung cancer patients and normal control patients. The VOCs were extracted from the headspace by solid-phase microextraction and were analyzed by gas chromatography time-of-flight mass spectrometry. Nine putative volatile biomarkers were identified as elevated in the lung cancer group. Receiver operating characteristic curve analysis was also performed, and the markers were found to be highly sensitive and specific. Next we used principal component analysis (PCA) modeling to make comparisons compare within the lung cancer group, and found that 2-pentanone may have utility in differentiating between adenocarcinoma and squamous cell carcinomas.

Analysis of volatile organic compounds released from human lung cancer cells and from the urine of tumor-bearing mice

BackgroundsA potential strategy for the diagnosis of lung cancer is to exploit the distinct metabolic signature of this disease by way of biomarkers found in different sample types. In this study, we investigated whether specific volatile organic compounds (VOCs) could be detected in the culture medium of the lung cancer cell line A549 in addition to the urine of mice implanted with A549 cells.ResultsSeveral VOCs were found at significantly increased or decreased concentrations in the headspace of the A549 cell culture medium as compared with the culture medium of two normal lung cell lines. We also analyzed the urine of mice implanted with A549 cells and several VOCs were also found to be significantly increased or decreased relative to urine obtained from control mice. It was also revealed that seven VOCs were found at increased concentrations in both sample types. These compounds were found to be dimethyl succinate, 2-pentanone, phenol, 2-methylpyrazine, 2-hexanone, 2-butanone and acetophenone.ConclusionsBoth sample types produce distinct biomarker profiles, and VOCs have potential to distinguish between true- and false-positive screens for lung cancer.

Asymmetrical distribution of the Schaffer projections within the apical dendrites of hippocampal field CA1.

Continuous current source densities were calculated in two dimensions (proximo-distal vs. medio-lateral) from slices of hippocampal field CA1 placed on a 64-electrode array in the presence of GABA blockers. The synaptic sink generated by stimulation of the Schaffer-commissural fibers spread across the extent of field CA1 within the same sublamina of the stratum radiatum as the stimulation electrode. The size and shape of the current sink varied according to the proximo-distal position of the stimulation site. Sinks generated by stimulation close to the cell body layer were more compact when compared to those produced by stimulation near the top of stratum radiatum which were broad and skewed in the proximal direction. These distributions were obtained with stimulation at either the CA3 or the subicular border of CA1. Induction of LTP increased the intensity of the current field but did not notably affect its distribution. It is concluded that (1) axons remain at the same proximo-distal level as they traverse stratum radiatum of CA1 and (2) generate proximally directed collaterals. Because of this, fibers that enter CA1 stratum radiatum immediately above the pyramidal cell bodies form compact synaptic fields while those entering CA1 at the top of the lamina form much broader and asymmetrical distributed fields.

Extracellular Recordings of Patterned Human Pluripotent Stem Cell-Derived Cardiomyocytes on Aligned Fibers

Human induced pluripotent stem cell (hiPSC) derived cardiomyocytes (CMs) hold high potential for use in drug assessment and myocardial regeneration. To create tissue-like constructs of CMs for extracellular monitoring, we placed aligned fibers (AFs) on the surface of a microelectrode array and then seeded hiPSC-CMs for subsequent monitoring for 14 days. As expected, the CMs organized into anisotropic and matured tissue and the extracellular recordings showed reduced premature beating higher signal amplitude and a higher probability of T-wave detection as compared to the culture without fibers. The CMs on the aligned fibers samples also exhibited anisotropic propagation of the field potential. These results therefore suggest that the hiPSC-CMs cultured on AFs can be used more reliably for cell based assays.

Deciphering the Receptor Repertoire Encoding Specific Odorants by Time-Lapse Single-Cell Array Cytometry

Mammals can recognize a vast number of odorants by using olfactory receptors (ORs) known as G protein-coupled receptors. The OR gene family is one of the most diverse gene families in mammalian genomes. Because of the vast combinations of ORs and odorants, few ORs have thus far been linked to specific odorants. Here, we established a functional screening method for OR genes by using a microchamber array containing >5,400 single olfactory epithelium-derived cells from mice applied to time-lapse single-cell array cytometry. This method facilitated the prompt isolation of single olfactory sensory neurons (OSNs) responding to the odorant of interest. Subsequent single-cell RT-PCR allowed us to isolate the genes encoding respective ORs. By using volatile molecules recognized as biomarkers for lung cancers, this method could deorphanize ORs and thereby reconstitute the OR-mediated signaling cascade in HEK293T cells. Thus, our system could be applied to identify any receptor by using specific ligands in the fields of physiopathology and pharmacology.

Quantitative and Single-step Enzyme Immunosensing Based on an Electrochemical Detection Coupled with Lateral-flow System

A single-step electrochemical immunochromatography has been developed: the device was based on two pieces of nitrocellulose membrane, a sample pad with anti-mouse IgG antibody labeled with glucose oxidase (GOx-labeled antibody), a conjugate pad with glucose, and a Pt working electrode. Either antibody or antigen was immobilized on the membrane. The addition of a solution containing mouse IgG, a model target, allows for the dissolution of GOx-labeled antibody in the sample pad to form an immunocomplex. The produced immunocomplex was automatically separated by capturing to the antibody immobilized on the membrane with the sandwich structure or by passing through the membrane modified with an antigen for the competitive reaction. The separated GOx label arrived at the conjugate pad with glucose to undergo the enzyme reaction. Hydrogen peroxide generated by this reaction was detected at the Pt electrode prepared on the second nitrocellulose membrane downstream from the conjugate pad. The results demonstrated that the designed immunochromatography can be applied to quantitative detection with a single-step procedure, because both the GOx-labeled antibody for revealing the immunoreactions and the substrate for the enzyme reaction were prepared in the device. Moreover, the initial concentration of the GOx-labeled antibody permitted control of the detectable concentration for mouse IgG.

Improvement of Electrochemical Response of Cocaine Sensors Based on DNA Aptamer by Heat Treatment

We report on a biosensor for cocaine based on the conformation change of DNA aptamer by capturing the cocaine molecules. The oxidation current of ferrocene conjugated on the terminal end of aptamer immobilized on an Au electrode increased with increasing cocaine concentration. The sensor response has been improved by a simple heat treatment after immobilization, since the aggregates of DNA aptamer generated during the immobilization step could be dissociated and rearranged on the electrode.