Dendritic spine generation and elimination play an important role in learning and memory, the dynamics of which have been examined within the neocortex in vivo. Spine turnover has also been detected in the absence of specific learning tasks, and is frequently exaggerated in animal models of autistic spectrum disorder (ASD). The present study aimed to examine whether the baseline rate of spine turnover was activity-dependent. This was achieved using a microfluidic brain interface and open-dura surgery, with the goal of abolishing neuronal Ca2+ signaling in the visual cortex of wild-type mice and rodent models of fragile X syndrome (Fmr1 knockout [KO]). In wild-type and Fmr1 KO mice, the majority of baseline turnover was found to be activity-independent. Accordingly, the application of matrix metalloproteinase-9 inhibitors selectively restored the abnormal spine dynamics observed in Fmr1 KO mice, without affecting the intrinsic dynamics of spine turnover in wild-type mice. Such findings indicate that the baseline turnover of dendritic spines is mediated by activity-independent intrinsic dynamics. Furthermore, these results suggest that the targeting of abnormal intrinsic dynamics might pose a novel therapy for ASD.

A miniaturized very-high-frequency- driven inductively coupled plasma jet source has been developed for the production of high-temperature and high-density plasmas in a small space, and its application to the localized and ultrahigh-rate etching of silicon wafers has been studied. The developed plasma source consists of a 1-mm-diam discharge tube with a fine nozzle of 0.1 mm in diameter at one end and a three-turn solenoidal antenna wound around it. The electron density of atmospheric-pressure argon plasma jets blowing out from the nozzle was estimated to be 1014–1015 cm−3 by means of optical emission spectroscopy. By the addition of halogen gases into the downstream region of argon plasma jets, high-speed etching of fine holes of several hundreds μm in diameter has been investigated. The highest etch rates of 4000 μm/min and 14 μm/min have been achieved for silicon wafers and fused silica glass wafers, respectively.

Phosphatidylserine (PS) has skewed distributions in the plasma membrane and is preferentially located in the inner leaflet of normal cells. Tumour cells, however, expose PS at the outer leaflet of cell surfaces, thereby potentially modulating the bio-signalling of cells. Interestingly, exosomes – or, more properly, small extracellular vesicles (sEVs) – which are secreted from tumour cells, are enriched with externalized PS, have been proposed as being involved in the progression of cancers, and could be used as a marker for tumour diagnostics. However, the sEV fractions prepared from various methods are composed of different subtypes of vesicles, and knowledge about the subtypes enriched with exposed PS is still limited. Here, we differentiated sEVs from cancer cell lines by density gradient centrifugation and characterized the separated fractions by using gold-labelling of PS in atomic force microscopy, thrombin generation assay, size and zeta potential measurements, and western blot analysis. These analyses revealed a previously unreported PS + -enriched sEV subtype, which is characterized by a lower density than that of canonical exosomes (1.06 g/ml vs. 1.08 g/ml), larger size (122 nm vs. 105 nm), more negative zeta potential (−28 mV vs. −21 mV), and lower abundance of canonical exosomal markers. The identification of the PS-exposed subtype of sEVs will provide deeper insight into the role of EVs in tumour biology and enhance the development of EV-based tumour diagnosis and therapy.

Extracellular vesicles (EVs) including exosomes and microvesicles have attracted considerable attention in the fields of cell biology and medicine. For a better understanding of EVs and further exploration of their applications, the development of analytical methods for biological nanovesicles has been required. In particular, considering the heterogeneity of EVs, methods capable of measuring individual vesicles are desired. Here, we report that on-chip immunoelectrophoresis can provide a useful method for the differential protein expression profiling of individual EVs. Electrophoresis experiments were performed on EVs collected from the culture supernatant of MDA-MB-231 human breast cancer cells using a measurement platform comprising a microcapillary electrophoresis chip and a laser dark-field microimaging system. The zeta potential distribution of EVs that reacted with an anti-human CD63 (exosome and microvesicle marker) antibody showed a marked positive shift as compared with that for the normal immunoglobulin G (IgG) isotype control. Thus, on-chip immunoelectrophoresis could sensitively detect the over-expression of CD63 glycoproteins on EVs. Moreover, to explore the applicability of on-chip immunoelectrophoresis to cancer diagnosis, EVs collected from the blood of a mouse tumor model were analyzed by this method. By comparing the zeta potential distributions of EVs after their immunochemical reaction with normal IgG, and the anti-human CD63 and anti-human CD44 (cancer stem cell marker) antibodies, EVs of tumor origin circulating in blood were differentially detected in the real sample. The result indicates that the present method is potentially applicable to liquid biopsy, a promising approach to the low-invasive diagnosis of cancer.

A miniaturized atmospheric-pressure thermal plasma jet source has been developed as a sensitive detector of a portable liquid analysis system that can fulfil various requirements of 'on-site' analysis. The plasma source design required for achieving higher power transfer efficiency to the plasma has been studied mainly so that it can be operated with a commercially available compact VHF transmitter. The developed plasma device is a planar-type inductively coupled plasma (ICP) source that consists of a ceramic chip with engraved discharge tube and a planar metallic antenna with serpentine structures. Effects of chip materials and antenna designs on plasma characteristics are discussed based on the result of optical diagnostics of fine argon plasma jets produced with various prototype sources. Since the load impedance of the plasma jet is so small, it is necessary to contrive ways to lower the antenna impedance to attain higher power transfer efficiency. An atmospheric-pressure thermal plasma jet with a density of approximately 1 × 10 15 cm −3 was successfully produced using a compact VHF transmitter at the 144 MHz VHF power of 50 W. The electronic excitation temperature of Ar was found to be 4000-4500 K. Moreover, the method of injection of the liquid sample into microplasma jets has also been investigated and preliminary results of the application to the analysis of tiny amounts of aqueous solution have been demonstrated.

Extracellular vesicles including exosomes have the potential to be used in therapeutic and diagnostic applications. Towards the noninvasive diagnosis of prostate cancer using urinary exosomes, technical challenges lie in developing precise methods of analyzing exosomes. Previously, we developed an on-chip microcapillary electrophoresis (µCE) system equipped with a laser dark-field microscope. The zeta potential of exosomes of cancer cells was found to be larger than that of normal cells. In this study, the zeta potential of exosomes of normal and cancer prostate cells was evaluated using this system after treating with sialidase. The large negative charge of cancer exosomes was found to be due to the large amount of sialic acids. These results suggest that an on-chip µCE system is useful for the accurate evaluation of events that occur on the exosome surfaces at the single-particle level and promising for the prescreening of prostate cancer exosomes without the need for labeling.

Studying cell functions for cellomics studies often requires the use of purified individual cells from mixtures of various kinds of cells. We have developed a new non-destructive on-chip cell sorting system for single cell based cultivation, by exploiting the advantage of microfluidics and electrostatic force. The system consists of the following two parts: a cell sorting chip made of polydimethylsiloxane (PDMS) on a 0.2-mm-thick glass slide, and an image analysis system with a phasecontrast/fluorescence microscope. The unique features of our system include (i) identification of a target from sample cells is achieved by comparison of the 0.2-µm-resolution phase-contrast and fluorescence images of cells in the microchannel every 1/30 s; (ii) non-destructive sorting of target cells in a laminar flow by application of electrostatic repulsion force for removing unrequited cells from the one laminar flow to the other; (iii) the use of agar gel for electrodes in order to minimize the effect on cells by electrochemical reactions of electrodes, and (iv) pre-filter, which was fabricated within the channel for removal of dust contained in a sample solution from tissue extracts. The sorting chip is capable of continuous operation and we have purified more than ten thousand cells for cultivation without damaging them. Our design has proved to be very efficient and suitable for the routine use in cell purification experiments.

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