Shohei Kaneda

Involvement of the Rabies Virus Phosphoprotein Gene in Neuroinvasiveness

ABSTRACT Rabies virus (RABV), which is transmitted via a bite wound caused by a rabid animal, infects peripheral nerves and then spreads to the central nervous system (CNS) before causing severe neurological symptoms and death in the infected individual. Despite the importance of this ability of the virus to spread from a peripheral site to the CNS (neuroinvasiveness) in the pathogenesis of rabies, little is known about the mechanism underlying the neuroinvasiveness of RABV. In this study, to obtain insights into the mechanism, we conducted comparative analysis of two fixed RABV strains, Nishigahara and the derivative strain Ni-CE, which cause lethal and asymptomatic infections, respectively, in mice after intramuscular inoculation. Examination of a series of chimeric viruses harboring the respective genes from Nishigahara in the genetic background of Ni-CE revealed that the Nishigahara phosphoprotein (P) gene plays a major role in the neuroinvasiveness by mediating infection of peripheral nerves. The results obtained from both in vivo and in vitro experiments strongly suggested that the Nishigahara P gene, but not the Ni-CE P gene, is important for stable viral replication in muscle cells. Further investigation based on the previous finding that RABV phosphoprotein counteracts the host interferon (IFN) system demonstrated that the Nishigahara P gene, but not the Ni-CE P gene, functions to suppress expression of the beta interferon (IFN-β) gene (Ifn-β) and IFN-stimulated genes in muscle cells. In conclusion, we provide the first data strongly suggesting that RABV phosphoprotein assists viral replication in muscle cells by counteracting the host IFN system and, consequently, enhances infection of peripheral nerves.

Cancer Cell Analyses at the Single Cell-Level Using Electroactive Microwell Array Device.

Circulating tumor cells (CTCs), shed from primary tumors and disseminated into peripheral blood, are playing a major role in metastasis. Even after isolation of CTCs from blood, the target cells are mixed with a population of other cell types. Here, we propose a new method for analyses of cell mixture at the single-cell level using a microfluidic device that contains arrayed electroactive microwells. Dielectrophoretic (DEP) force, induced by the electrodes patterned on the bottom surface of the microwells, allows efficient trapping and stable positioning of single cells for high-throughput biochemical analyses. We demonstrated that various on-chip analyses including immunostaining, viability/apoptosis assay and fluorescent in situ hybridization (FISH) at the single-cell level could be conducted just by applying specific reagents for each assay. Our simple method should greatly help discrimination and analysis of rare cancer cells among a population of blood cells.

Generation of a Motor Nerve Organoid with Human Stem Cell-Derived Neurons

Summary During development, axons spontaneously assemble into a fascicle to form nerves and tracts in the nervous system as they extend within a spatially constrained path. However, understanding of the axonal fascicle has been hampered by lack of an in vitro model system. Here, we report generation of a nerve organoid composed of a robust fascicle of axons extended from a spheroid of human stem cell-derived motor neurons within our custom-designed microdevice. The device is equipped with a narrow channel providing a microenvironment that facilitates the growing axons to spontaneously assemble into a unidirectional fascicle. The fascicle was specifically made with axons. We found that it was electrically active and elastic and could serve as a model to evaluate degeneration of axons in vitro. This nerve organoid model should facilitate future studies on the development of the axonal fascicle and drug screening for diseases affecting axon fascicles.

Single-step CE for miniaturized and easy-to-use system.

We developed a novel single‐step capillary electrophoresis (SSCE) scheme for miniaturized and easy to use system by using a microchannel chip, which was made from the hydrophilic material polymethyl methacrylate (PMMA), equipped with a capillary stop valve. Taking the surface tension property of liquids into consideration, the capillary effect was used to introduce liquids and control capillary stop valves in a partial barrier structure in the wall of the microchannel. Through the combined action of stop valves and air vents, both sample plug formation for electrophoresis and sample injection into a separation channel were successfully performed in a single step. To optimize SSCE, different stop valve structures were evaluated using actual microchannel chips and the finite element method with the level set method. A partial barrier structure at the bottom of the channel functioned efficiently as a stop valve. The stability of stop valve was confirmed by a shock test, which was performed by dropping the microchannel chip to a floor. Sample plug deformation could be reduced by minimizing the size of the side partial barrier. By dissolving hydroxyl ethyl cellulose and using it as the sample solution, the EOF and adsorption of the sample into the PMMA microchannel were successfully reduced. Using this method, a 100‐bp DNA ladder was concentrated; good separation was observed within 1 min. At a separation length of 5 mm, the signal was approximately 20‐fold higher than a signal of original sample solution by field‐amplified sample stacking effect. All operations, including liquid introduction and sample separation, can be completed within 2 min by using the SSCE scheme.

Integrated in situ genetic analyzer for microbiology in extreme environments

In this study, a totally integrated in situ analyzer for microbial gene detection has been developed for oceanography applications. A PDMS–glass microfluidic device that is capable of cell lysis, DNA purification, PCR, and optical detection has been utilized as the core element of the in situ analyzer. Microbial genomic DNA is purified and concentrated on glass beads packed in the microfluidic device. PCR is performed in a flow-through manner, and the amplified products are fluorescently detected using optical fibers. The sensitivity of a completed analyzer with deep-sea operation capabilities has been evaluated in a laboratory setting, and the analyzer has been operated in real deep-sea environments. Field evaluations have shown that the amplification of the eubacterial universal 16S rRNA gene and the recovery of the PCR product to the surface are successfully achieved in deep-sea environments.

Pneumatic handling of droplets on-demand on a microfluidic device for seamless processing of reaction and electrophoretic separation

Sequential operations of pre‐separation reaction process by picoliter droplets and following electrophoretic separation process were realized in a single microfluidic device with pneumatic handling of liquid. The developed device consists of a fluidic chip made of PDMS, an electrode substrate, and a temperature control substrate on which thin film heater/sensor structures are fabricated. Liquid handling, including introduction of liquid samples, droplet generation, and merging of droplets, was implemented by pneumatic manipulation through microcapillary vent structures, allowing air to pass and stop liquid flow. Since the pneumatic manipulations are conducted in a fully automated manner by using a programmable air pressure control system, the user simply has to load liquid samples on each liquid port of the device. Droplets of 420 pL were generated with an accuracy of ±2 pL by applying droplet generation pressure in the range of 40–100 kPa. As a demonstration, a binding reaction of a 15mer ssDNA with a peptide nucleic acid oligomer used as an oligoprobe followed by denaturing electrophoresis to discriminate a single‐base substitution was performed within 1.5 min. By exploiting the droplet‐on‐demand capability of the device, the influence of various factors, such as reaction time, mixing ratio and droplet configurations on the ssDNA‐peptide nucleic acid binding reaction in the droplet‐based process, was studied toward realization of a rapid detection method to discriminate rapid single‐base substitution.

Optofluidic tweezer on a chip

A novel method to realize an optical tweezer involving optofluidic operation in a microchannel is proposed. To manipulate the optical tweezer, light from an optical fiber is passed through both PDMS (polydimethylsiloxane)-air surface lenses and an optofluidic region, which is located in a control channel. Two liquids with different refractive indices (RIs) are introduced into the control channel to form two different flow patterns (i.e., laminar and segmented flows), depending on the liquid compositions, the channel geometry, and the flow rates. By altering the shapes of the interface of the two liquids in the optofluidic region, we can continuously or intermittently control the optical paths of the light. To demonstrate the functionality of the proposed method, optical tweezer operations on a chip are performed. Changing the flow pattern of two liquids with different RIs in the optofluidic region results in successful trapping of a 25 μm diameter microsphere and its displacement by 15 μm.

Integrated Microfluidic Systems

Using unique physical phenomena at the microscale, such as laminar flow, mixing by diffusion, relative increase of the efficiency of heat exchange, surface tension and friction due to the increase of surface-to-volume ratio by downscaling, research in the field of microfluidic devices, aims at miniaturization of (bio)chemical apparatus for high-throughput analyses. Microchannel networks as core components of microfluidic devices are fabricated on various materials, such as silicon, glass, polymers, metals, etc., using microfabrication techniques adopted from the semiconductor industry and microelectromechanical systems (MEMS) technology, enabling integration of the components capable of performing various operations in microchannel networks. This chapter describes examples of diverse integrated microfluidic devices that incorporate functional components such as heaters for reaction temperature control, micropumps for liquid transportation, air vent structures for pneumatic manipulation of small volume droplets, optical fibers with aspherical lens structures for fluorescence detection, and electrochemical sensors for monitoring of glucose consumption during cell culture. The focus of this review is these integrated components and systems that realize useful functionalities for biochemical analyses.

Application of cell-free expression of GFP for evaluation of microsystems.

Coupled cell-free transcription-translation (CFTT) of green fluorescent protein (GFP) has been applied as a reporter system to microfluidic chip-related technologies. In polymerase chain reaction (PCR)-based biomolecular logic gate system, in which addition of primer set and amplification of PCR product represent input and output signal respectively, GFP gene was inserted in the template DNA, which was then amplified, transcribed and translated to GFP. The green fluorescence reported as if the amplification has occurred or not, that is, the fluorescence reports positive output signal. CFTT of GFP was also adopted to evaluate on-chip capillary electrophoresis (CE)-based DNA fractionation, which was developed to isolate single DNA species from reaction mixture of DNA ligase-catalyzed DNA-assembly. As a model system, GFP gene was inserted in the target DNA fragment. The collected fraction was amplified with PCR and subjected to a CFTT system, and green fluorescence was observed showing that the fractionation was successful. These results showed that CFTT of GFP is a useful tool to verify, estimate, and monitor microfluidic chip-related technologies in which cell-free protein synthesis is involved.

A Rapid Method for Optimizing Running Temperature of Electrophoresis through Repetitive On-Chip CE Operations

In this paper, a rapid and simple method to determine the optimal temperature conditions for denaturant electrophoresis using a temperature-controlled on-chip capillary electrophoresis (CE) device is presented. Since on-chip CE operations including sample loading, injection and separation are carried out just by switching the electric field, we can repeat consecutive run-to-run CE operations on a single on-chip CE device by programming the voltage sequences. By utilizing the high-speed separation and the repeatability of the on-chip CE, a series of electrophoretic operations with different running temperatures can be implemented. Using separations of reaction products of single-stranded DNA (ssDNA) with a peptide nucleic acid (PNA) oligomer, the effectiveness of the presented method to determine the optimal temperature conditions required to discriminate a single-base substitution (SBS) between two different ssDNAs is demonstrated. It is shown that a single run for one temperature condition can be executed within 4 min, and the optimal temperature to discriminate the SBS could be successfully found using the present method.