Analyzing threshold pressure limitations in microfluidic transistors for self-regulated microfluidic circuits

This paper reveals a critical limitation in the electro-hydraulic analogy between a microfluidic membrane-valve (mu MV) and an electronic transistor. Unlike typical transistors that have similar on and off threshold voltages, in hydraulic mu MVs, the threshold pressures for opening and closing are significantly different and can change, even for the same mu MVs depending on overall circuit design and operation conditions. We explain, in particular, how the negative values of the closing threshold pressures significantly constrain operation of even simple hydraulic mu MV circuits such as autonomously switching two-valve microfluidic oscillators. These understandings have significant implications in designing self-regulated microfluidic devicesopen4

Growth rate-dependent flexural rigidity of microtubules influences pattern formation in collective motion

[Background] Microtubules (MTs) are highly dynamic tubular cytoskeleton filaments that are essential for cellular morphology and intracellular transport. In vivo, the flexural rigidity of MTs can be dynamically regulated depending on their intracellular function. In the in vitro reconstructed MT-motor system, flexural rigidity affects MT gliding behaviors and trajectories. Despite the importance of flexural rigidity for both biological functions and in vitro applications, there is no clear interpretation of the regulation of MT flexural rigidity, and the results of many studies are contradictory. These discrepancies impede our understanding of the regulation of MT flexural rigidity, thereby challenging its precise manipulation. [Results] Here, plausible explanations for these discrepancies are provided and a new method to evaluate the MT rigidity is developed. Moreover, a new relationship of the dynamic and mechanic of MTs is revealed that MT flexural rigidity decreases through three phases with the growth rate increases, which offers a method of designing MT flexural rigidity by regulating its growth rate. To test the validity of this method, the gliding performances of MTs with different flexural rigidities polymerized at different growth rates are examined. The growth rate-dependent flexural rigidity of MTs is experimentally found to influence the pattern formation in collective motion using gliding motility assay, which is further validated using machine learning. [Conclusion] Our study establishes a robust quantitative method for measurement and design of MT flexural rigidity to study its influences on MT gliding assays, collective motion, and other biological activities in vitro. The new relationship about the growth rate and rigidity of MTs updates current concepts on the dynamics and mechanics of MTs and provides comparable data for investigating the regulation mechanism of MT rigidity in vivo in the future

Long-term effect of sodium selenite on the integrity and permeability of on-chip microvasculature

Development of the robust and functionally stable three-dimensional (3D) microvasculature remains challenging. One often-overlooked factor is the presence of potential anti-angiogenic agents in culture media. Sodium selenite, an antioxidant commonly used in serum-free media, demonstrates strong anti-angiogenic properties and has been proposed as an anticancer drug. However, its long-term effects on in vitro microvascular systems at the concentrations used in culture media have not been studied. In this study, we used a five-channel microfluidic device to investigate the concentration and temporal effects of sodium selenite on the morphology and functionality of on-chip preformed microvasculature. We found that high concentrations (∼3.0 μM) had adverse effects on microvasculature perfusion, permeability, and overall integrity within the first few days. Moreover, even at low concentrations (∼3.0 nM), a long-term culture effect was observed, resulting in an increase in vascular permeability without any noticeable changes in morphology. A further analysis suggested that vessel leakage may be due to vascular endothelial growth factor dysregulation, disruption of intracellular junctions, or both. This study provides important insight into the adverse effects caused by the routinely present sodium selenite on 3D microvasculature in long-term studies for its application in disease modeling and drug screening

A new perfusion culture method with a self-organized capillary network

A lack of perfusion has been one of the most significant obstacles for three-dimensional culture systems of organoids and embryonic tissues. Here, we developed a simple and reliable method to implement a perfusable capillary network in vitro. The method employed the self-organization of endothelial cells to generate a capillary network and a static pressure difference for culture medium circulation, which can be easily introduced to standard biological laboratories and enables long-term cultivation of vascular structures. Using this culture system, we perfused the lumen of the self-organized capillary network and observed a flow-induced vascular remodeling process, cell shape changes, and collective cell migration. We also observed an increase in cell proliferation around the self-organized vasculature induced by flow, indicating functional perfusion of the culture medium. We also reconstructed extravasation of tumor and inflammatory cells, and circulation inside spheroids including endothelial cells and human lung fibroblasts. In conclusion, this system is a promising tool to elucidate the mechanisms of various biological processes related to vascular flow

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

HapBead: on-skin microfluidic haptic interface using tunable bead

On-skin haptic interfaces using soft elastomers which are thin and flexible have significantly improved in recent years. Many are focused on vibrotactile feedback that requires complicated parameter tuning. Another approach is based on mechanical forces created via piezoelectric devices and other methods for non-vibratory haptic sensations like stretching, twisting. These are often bulky with electronic components and associated drivers are complicated with limited control of timing and precision. This paper proposes HapBead, a new on-skin haptic interface that is capable of rendering vibration like tactile feedback using microfluidics. HapBead leverages a microfluidic channel to precisely and agilely oscillate a small bead via liquid flow, which then generates various motion patterns in channel that creates highly tunable haptic sensations on skin. We developed a proof-of-concept design to implement thin, flexible and easily affordable HapBead platform, and verified its haptic rendering capabilities via attaching it to users’ fingertips. A study was carried out and confirmed that participants could accurately tell six different haptic patterns rendered by HapBead. HapBead enables new wearable display applications with multiple integrated functionalities such as on-skin haptic doodles, mixed reality haptics and visual-haptic displays

Linear-Zero Mode Waveguides for Single-Molecule Fluorescence Observation of Nucleotides in Kinesin-Microtubule Motility Assay

Part of the Methods in Molecular Biology book series (MIMB, volume 2430)Single-molecule fluorescence microscopy is a key tool to investigate the chemo-mechanical coupling of microtubule-associated motor proteins, such as kinesin. However, a major limitation of the implementation of single-molecule observation is the concentration of fluorescently labeled molecules. For example, in total internal reflection fluorescence microscopy, the available concentration is of the order of 10 nM. This concentration is much lower than the concentration of adenosine triphosphate (ATP) in vivo, hindering the single-molecule observation of fluorescently labeled ATP hydrolyzed by motor proteins under the physiologically relevant conditions. Here, we provide a method for the use of single-molecule fluorescence microscopy in the presence of ~500 nM of fluorescently labeled ATP. To achieve this, a device equipped with nano-slits is used to confine excitation light into its slits as an expansion of zero-mode waveguides (ZMWs). Conventional ZMWs equip apertures with a diameter smaller than the wavelength of light to suppress background noise from the labeled molecules diffusing outside of the apertures. While they are not compatible with filamentous objects, our linear-ZMWs enable the usage of filamentous objects, such as microtubules. An experiment using linear-ZMWs demonstrated the successful exploration of the interaction between kinesin and ATP using single-molecule fluorescence microscopy

Piezoelectric properties of microfabricated (K,Na)NbO3 thin films

A novel microfabrication method of lead-free piezoelectric sodium potassium niobate [(K, Na)NbO3, KNN] thin films was proposed, and the piezoelectric characteristics of the KNN microactuators were evaluated. The KNN thin films were directly deposited on microfabricated Si microcantilevers. The transverse piezoelectric coefficient d31 of the KNN films was calculated as −53.5 pm/V at 20 Vpp from the tip displacement of the microcantilevers. However, the tip displacement showed large electric-field dependence because of the extrinsic piezoelectric effect, and the intrinsic piezoelectric effect of the KNN microcantilevers was smaller than that of KNN on unprocessed thick substrates. In contrast, the extrinsic piezoelectric effect was almost independent of the microfabrication of the KNN films

Different motilities of microtubules driven by kinesin-1 and kinesin-14 motors patterned on nanopillars

モータータンパク質は種類により協働性が異なることを発見 --分子を自在に並べる技術により生体分子モーターの協働性を計測--. 京都大学プレスリリース. 2020-01-23.How're your cells' motors running?. 京都大学プレスリリース. 2020-01-23.Kinesin is a motor protein that plays important roles in a variety of cellular functions. In vivo, multiple kinesin molecules are bound to cargo and work as a team to produce larger forces or higher speeds than a single kinesin. However, the coordination of kinesins remains poorly understood because of the experimental difficulty in controlling the number and arrangement of kinesins, which are considered to affect their coordination. Here, we report that both the number and spacing significantly influence the velocity of microtubules driven by nonprocessive kinesin-14 (Ncd), whereas neither the number nor the spacing changes the velocity in the case of highly processive kinesin-1. This result was realized by the optimum nanopatterning method of kinesins that enables immobilization of a single kinesin on a nanopillar. Our proposed method enables us to study the individual effects of the number and spacing of motors on the collective dynamics of multiple motors

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

「分子綱引き」をおこなうナノシステムを開発 -細胞分裂や細胞内物質輸送の仕組みを知るカギに-.京都大学プレスリリース. 2014-06-17.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