Hirofumi Shintaku

Piezoelectric materials mimic the function of the cochlear sensory epithelium

Cochlear hair cells convert sound vibration into electrical potential, and loss of these cells diminishes auditory function. In response to mechanical stimuli, piezoelectric materials generate electricity, suggesting that they could be used in place of hair cells to create an artificial cochlear epithelium. Here, we report that a piezoelectric membrane generated electrical potentials in response to sound stimuli that were able to induce auditory brainstem responses in deafened guinea pigs, indicating its capacity to mimic basilar membrane function. In addition, sound stimuli were transmitted through the external auditory canal to a piezoelectric membrane implanted in the cochlea, inducing it to vibrate. The application of sound to the middle ear ossicle induced voltage output from the implanted piezoelectric membrane. These findings establish the fundamental principles for the development of hearing devices using piezoelectric materials, although there are many problems to be overcome before practical application.

Development of coarse-graining DNA models for single-nucleotide resolution analysis

Recently, analytical techniques have been developed for detecting single-nucleotide polymorphisms in DNA sequences. Improvements of the sequence identification techniques has attracted much attention in several fields. However, there are many things that have not been clarified about DNA. In the present study, we have developed a coarse-graining DNA model with single-nucleotide resolution, in which potential functions for hydrogen bonds and the π-stack effect are taken into account. Using Langevin-dynamics simulations, several characteristics of the coarse-grained DNA have been clarified. The validity of the present model has been confirmed, compared with other experimental and computational results. In particular, the melting temperature and persistence length are in good agreement with the experimental results for a wide range of salt concentrations.

On-Chip Separation and Analysis of RNA and DNA from Single Cells

The simultaneous analysis of RNA and DNA of single cells remains a challenge as these species have very similar physical and biochemical properties and can cross-contaminate each other. Presented is an on-chip system that enables selective lysing of single living cells, extraction, focusing, and absolute quantification of cytoplasmic RNA mass and its physical separation from DNA in the nucleus using electrical lysing and isotachophoresis (ITP). This absolute quantitation is performed without enzymatic amplification in less than 5 min. The nucleus is preserved, and its DNA fluorescence signal can be measured independently. We demonstrate the technique using single mouse lymphocyte cells, for which we extracted an average of 14.1 pg of total RNA per cell. We also demonstrate correlation analysis between the absolute amount of RNA and relative amount of DNA, showing heterogeneity associated with cell cycles. The technique is compatible with fractionation of DNA and RNA and with downstream assays of each.

Effects of fluid dynamic stress on fracturing of cell-aggregated tissue during purification for islets of Langerhans transplantation

Among clinical treatments for type 1 diabetes mellitus, the transplantation of islets of Langerhans to the portal vein of the hepar is a commonly used treatment for glucose homeostasis. Islet purification using the density gradient of a solution in a centrifuge separator is required for safety and efficiency. In the purification, the number of tissues to be transplanted is reduced by removing the acinar tissue and gathering the islet from the digest of pancreas. However, the mechanical effects on the fracture of islets in the centrifuge due to fluid dynamic stress are a serious problem in the purification process. In this study, a preliminary experiment using a cylindrical rotating viscometer with a simple geometry is conducted in order to systematically clarify the effect of fluid dynamic stress on the fracture of islets. The effects of fluid dynamic stress on the islet configuration is quantitatively measured for various flow conditions, and a predictive fracture model is developed based on the experimental results. Furthermore, in the practical purification process in the COBE (Gambro BCT), which is widely used in clinical applications, we perform a numerical analysis of the fluid dynamic stress based on Navier–Stokes equations to estimate the stress conditions for islets. Using the fracture model and numerical analysis, the islet fracture characteristics using the COBE are successfully investigated. The results obtained in this study provide crucial information for the purification of islets by centrifuge in practical and clinical applications.

Wide-range frequency selectivity in an acoustic sensor fabricated using a microbeam array with non-uniform thickness

In this study, we have demonstrated the fabrication of a microbeam array (MBA) with various thicknesses and investigated the suitability it for an acoustic sensor with wide-range frequency selectivity. For this, an MBA composed of 64 beams, with thicknesses varying from 2.99–142 µm, was fabricated by using single gray-scale lithography and a thick negative photoresist. The vibration of the beams in air was measured using a laser Doppler vibrometer; the resonant frequencies of the beams were measured to be from 11.5 to 290 kHz. Lastly, the frequency range of the MBA with non-uniform thickness was 10.9 times that of the MBA with uniform thickness.

Control of microtubule trajectory within an electric field by altering surface charge density

One of challenges for using microtubules (MTs) driven by kinesin motors in microfluidic environments is to control their direction of movement. Although applying physical biases to rectify MTs is prevalent, it has not been established as a design methodology in conjunction with microfluidic devices. In the future, the methodology is expected to achieve functional motor-driven nanosystems. Here, we propose a method to guide kinesin-propelled MTs in multiple directions under an electric field by designing a charged surface of MT minus ends labeled with dsDNA via a streptavidin-biotin interaction. MTs labeled with 20-bp or 50-bp dsDNA molecules showed significantly different trajectories according to the DNA length, which were in good agreement with values predicted from electrophoretic mobilities measured for their minus ends. Since the effective charge of labeled DNA molecules was equal to that of freely dispersed DNA molecules in a buffer solution, MT trajectory could be estimated by selecting labeling molecules with known charges. Moreover, the estimated trajectory enables to define geometrical sizes of a microfluidic device. This rational molecular design and prediction methodology allows MTs to be guided in multiple directions, demonstrating the feasibility of using molecular sorters driven by motor proteins.

Tracking single-particle dynamics via combined optical and electrical sensing.

While fluorescent imaging has been extensively used for single-particle tracking, temporal and spatial resolution of the wide-field microscopy technology is not satisfactory for investigating fast-moving features. Here we report a method for probing nanometer-scale motion of an individual particle through a microstructured channel at sub-MHz by combining a resistive pulse technique to the optical sensing. We demonstrate unambiguous discriminations of translocation and non-translocation events inferred from spike-like electrical signals by fluorescence images captured synchronously to ionic current measurements. We also find a trajectory-dependent translocation dynamics of voltage-driven single-particles through a microchannel. This electrical/optical approach may find applications in sensor technologies based on micro- and nano-electromechanical systems.

A hardware model of the auditory periphery to transduce acoustic signals into neural activity.

To improve the performance of cochlear implants, we have integrated a microdevice into a model of the auditory periphery with the goal of creating a microprocessor. We constructed an artificial peripheral auditory system using a hybrid model in which polyvinylidene difluoride was used as a piezoelectric sensor to convert mechanical stimuli into electric signals. To produce frequency selectivity, the slit on a stainless steel base plate was designed such that the local resonance frequency of the membrane over the slit reflected the transfer function. In the acoustic sensor, electric signals were generated based on the piezoelectric effect from local stress in the membrane. The electrodes on the resonating plate produced relatively large electric output signals. The signals were fed into a computer model that mimicked some functions of inner hair cells, inner hair cell–auditory nerve synapses, and auditory nerve fibers. In general, the responses of the model to pure-tone burst and complex stimuli accurately represented the discharge rates of high-spontaneous-rate auditory nerve fibers across a range of frequencies greater than 1 kHz and middle to high sound pressure levels. Thus, the model provides a tool to understand information processing in the peripheral auditory system and a basic design for connecting artificial acoustic sensors to the peripheral auditory nervous system. Finally, we discuss the need for stimulus control with an appropriate model of the auditory periphery based on auditory brainstem responses that were electrically evoked by different temporal pulse patterns with the same pulse number.

Tug-of-war of microtubule filaments at the boundary of a kinesin- and dynein-patterned surface.

Intracellular cargo is transported by multiple motor proteins. Because of the force balance of motors with mixed polarities, cargo moves bidirectionally to achieve biological functions. Here, we propose a microtubule gliding assay for a tug-of-war study of kinesin and dynein. A boundary of the two motor groups is created by photolithographically patterning gold to selectively attach kinesin to the glass and dynein to the gold surface using a self-assembled monolayer. The relationship between the ratio of two antagonistic motor numbers and the velocity is derived from a force-velocity relationship for each motor to calculate the detachment force and motor backward velocity. Although the tug-of-war involves >100 motors, values are calculated for a single molecule and reflect the collective dynein and non-collective kinesin functions when they work as a team. This assay would be useful for detailed in vitro analysis of intracellular motility, e.g., mitosis, where a large number of motors with mixed polarities are involved.

SINC-seq: correlation of transient gene expressions between nucleus and cytoplasm reflects single-cell physiology

We report a microfluidic system that physically separates nuclear RNA (nucRNA) and cytoplasmic RNA (cytRNA) from a single cell and enables single-cell integrated nucRNA and cytRNA-sequencing (SINC-seq). SINC-seq constructs two individual RNA-seq libraries, nucRNA and cytRNA, per cell, quantifies gene expression in the subcellular compartments, and combines them to create novel single-cell RNA-seq data. Leveraging SINC-seq, we discover distinct natures of correlation among cytRNA and nucRNA that reflect the transient physiological state of single cells. These data provide unique insights into the regulatory network of messenger RNA from the nucleus toward the cytoplasm at the single-cell level.