Morphological and functional analyses for investigation of sexually selected legs in the frog legged beetle Sagra femorata (Coleoptera: Chrysomelidae).

Mate choice and male-male combat over successful mating often cause disproportionate exaggeration of male trait relative to body size. However, the exaggeration is often not the only trait involved with male-male combat and mate choice: suites of co-expressed traits may function together as a coordinated unit. When this occurs, dimorphism may be expected for these additional, non-exaggerated, structures. S. femorata males have disproportionately large hind-legs used in male-male combat over females. During the fights, fore- and mid-legs are used to keep males in positions where advantageous for leverage. Because use of the exaggerated hind-legs is coordinated with the other legs, they will coevolve as a functional unit. Here, we show that 1) S. femorata has sexual size differences in all three legs; 2) males show positive allometry in the relative sizes of all three legs; and 3) microstructures of tarsi on the fore- and mid-legs are also sexually dimorphic. Despite these differences in the tarsal microstructure, 4) adhesion forces of the tarsi had no sexual difference in flat surface. The microstructure would be specialized on attaching elytra surface. These results suggest that the three pairs of legs function together during fighting behavior, with hind-legs employed primarily for fighting, and the fore- and mid-legs functioning to grip females, keeping males positioned on the back of the female during combat.

Micro tensile tester measurement of biomechanical properties and adhesion force of microtubule-polymerization-inhibited cancer cells.

Both mechanical and adhesion properties of cancer cells are complex and reciprocally related to migration, invasion, and metastasis with large cell deformation. Therefore, we evaluated these properties for human cervical cancer cells (HeLa) simultaneously using our previously developed micro tensile tester system. For efficient evaluation, we developed image analysis software to modify the system. The software can analyze the tensile force in real time. The modified system can evaluate the tensile stiffness of cells to which a large deformation is applied, also evaluate the adhesion strength of cancer cells that adhered to a culture substrate and were cultured for several days with their adhesion maturation. We used the modified system to simultaneously evaluate the stiffness of the cancer cells to which a large deformation was applied and their adhesion strength. The obtained results revealed that the middle phase of tensile stiffness and adhesion force of the microtubule-depolymerized group treated with colchicine (an anti-cancer drug) (stiffness, 13.4 ± 7.5 nN/%; adhesion force, 460.6 ± 258.2 nN) were over two times larger than those of the control group (stiffness, 5.0 ± 3.5 nN/%; adhesion force, 168.2 ± 98.0 nN). Additionally, the same trend was confirmed with the detailed evaluation of cell surface stiffness using an atomic force microscope. Confocal fluorescence microscope observations showed that the stress fibers (SFs) of colchicine-treated cells were aligned in the same direction, and focal adhesions (FAs) of the cells developed around both ends of the SFs and aligned parallel to the developed direction of the SFs. There was a possibility that the microtubule depolymerization by the colchicine treatment induced the development of SFs and FAs and subsequently caused an increment of cell stiffness and adhesion force. From the above results, we concluded the modified system would be applicable to cancer detection and anti-cancer drug efficacy tests.

Contractile and Tensile Measurement of Molecular Artificial Muscles for Biohybrid Robotics.

A printable artificial muscle assembled from biomolecular motors, which we have recently developed, showed great potential in overcoming the design limitations of conventional biohybrid robots as a new bio-actuator. Characterizing its contractility for extending its applicability is important. However, conventional measurement methods are composed of complex operations with poor reproducibility, flexibility, and real-time responsiveness. This study presents a new method for measuring the contractile force generated by artificial muscles. A measurement system was constructed, wherein artificial muscles were patterned by UV laser scanning in an oil-sealed microchamber, and the contractile force was measured in real time using a microforce sensor extended by a 3D-printed microcantilever. The measurement accuracy of the sensor was ensured through calibration and correction. For demonstration purposes, a series of contractile measurements were carried out using the proposed system. The relationship between contractile force and the dimensions of the activation space of the artificial muscles, as well as the tensile properties of the contracted muscle chain were evaluated. The results will help characterize the contractile properties of the artificial muscle and lay the foundations for its further application in biohybrid robotics.

Analysis of Rowing Force of the Water Strider Middle Leg by Direct Measurement Using a Bio-Appropriating Probe and by Indirect Measurement Using Image Analysis.

Rowing force of the middle leg of a water strider is one of the important factors affecting water repellency and applications in biomimetics, biomechanics, and biology. However, many previous studies have been based on estimated leg rowing force and lack some credibility. Therefore, we tried to measure leg rowing force directly by a force transducer. In this article, we report the rowing force of water striders obtained by direct and indirect measurements. In the direct measurement, water striders were set onto a sensor system and the rowing force of a middle leg of the set water striders was directly measured using a bio-appropriating probe (BAP), a kind of hook. In the indirect measurement, water striders were not fixed and the rowing force of locomoting water striders was evaluated by image analysis using a high-speed camera. As a result, we determined the rowing force by the direct measurement to be 955 µN, while the rowing force by the indirect measurement was 493 µN. We considered that the indirect measurement might lack some credibility because half the propellant energy was lost in the indirect force measurement due to various other factors.

Survival Rate of Cells Sent by a Low Mechanical Load Tube Pump: The "Ring Pump".

A ring pump (RP) is a useful tool for microchannels and automated cell culturing. We have been developing RPs (a full-press ring pump, FRP; and a mid-press ring pump, MRP). However, damage to cells which were sent by the RP and the MRP was not investigated, and no other studies have compared the damage to cells between RPs and peristaltic pumps (PPs). Therefore, first, we evaluated the damage to cells that were sent by a small size FRP (s-FRP) and small size MRPs (s-MRPs; gap = 25 or 50 µm, respectively). "Small size" means that the s-FRP and the s-MRPs are suitable for microchannel-scale applications. The survival rate of cells sent by the s-MRPs was higher than those sent by the s-FRP, and less damage caused by the former. Second, we compared the survival rate of cells that were sent by a large size FRP (l-FRP), a large size MRP (l-MRP) (gap = 50 µm) and a PP. "Large size" means that the l-FRP and the l-MRP are suitable for automated cell culture system applications. We could not confirm any differences among the cell survival rates. On the other hand, when cells suspended in Dulbecco's phosphate-buffered saline solution were circulated with the l-MRP (gap = 50 µm) and the PP, we confirmed a difference in cell survival rate, and less damage caused by the former.

Micro Vacuum Chuck and Tensile Test System for Bio-Mechanical Evaluation of 3D Tissue Constructed of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes (hiPS-CM).

In this report, we propose a micro vacuum chuck (MVC) which can connect three-dimensional (3D) tissues to a tensile test system by vacuum pressure. Because the MVC fixes the 3D tissue by vacuum pressure generated on multiple vacuum holes, it is expected that the MVC can fix 3D tissue to the system easily and mitigate the damage which can happen by handling during fixing. In order to decide optimum conditions for the size of the vacuum holes and the vacuum pressure, various sized vacuum holes and vacuum pressures were applied to a normal human cardiac fibroblast 3D tissue. From the results, we confirmed that a square shape with 100 µm sides was better for fixing the 3D tissue. Then we mounted our developed MVCs on a specially developed tensile test system and measured the bio-mechanical property (beating force) of cardiac 3D tissue which was constructed of human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CM); the 3D tissue had been assembled by the layer-by-layer (LbL) method. We measured the beating force of the cardiac 3D tissue and confirmed the measured force followed the Frank-Starling relationship. This indicates that the beating property of cardiac 3D tissue obtained by the LbL method was close to that of native cardiac tissue.

Analytical Model and Experimental Evaluation of the Micro-Scale Thermal Property Sensor for Single-Sided Measurement.

We report a new analytical model of the MEMS-based thermal property sensor for samples which are difficult to handle and susceptible to damage by thermal stimulus, such as living cells. Many sensor designs had been reported for thermal property measurements, but only a few of them have considered the analytical model of the single-sided measurement in which a measurement sample is placed on the sensor substrate. Even in the few designs that have considered the analytical model, their applicable limits are restricted to more than 1 mm length in practical situations. Our new model considers both the sample and the sensor substrate thermal properties and is applicable to a sensor length less than 1 µm. In order to minimize the influence of the heat stimulus to the sample, the model formulates the required heat dissipating time for different sensor geometries. We propose fast and precise detection circuit architecture to realize our model, and we discuss the sensor performance for a number of different designs.

A Closed System for Pico-Liter Order Substance Transport from a Giant Liposome to a Cell.

In single cell analysis, transport of foreign substances into a cell is an important technique. In particular, for accurate analysis, a method to transport a small amount (pico-liter order) of substance into the cell without leakage while retaining the cell shape is essential. Because the fusion of the cell and the giant liposome is a closed system to the outside, it may be possible to transport a precise, small amount of substances into the cell. Additionally, there is no possibility that a leaked substance would affect other systems. To develop the liposome-cell transportation system, knowledge about the behavior of substances in the liposome and the cell is important. However, only a few studies have observed the substance transport between a liposome and a cell. Here, we report observation of small amount of substance transport into a single C2C12 cell by using a giant liposome. Substance transport occurred by electrofusion between the cell and the giant liposome containing the substance, which is a closed system. First, to observe the electrofusion and substance transport from the moment of voltage application, we fabricated a microfluidic device equipped with electrodes. We introduced suspensions of cells and liposomes into the microfluidic device and applied alternating current (AC) and direct current (DC) voltages for electrofusion. We observed a small amount (22.4 ± 0.1%, 10.3 ± 0.4% and 9.1 ± 0.1%) of fluorescent substance (Calcein) contained in the liposomes was transported into the cell without leakage outside the cell, and we obtained the diffusion coefficient of Calcein in the cell as 137 ± 18 µm²/s. We anticipate that this system and the knowledge acquired will contribute to future realization of more accurate single cell analysis in a wide range of fields.