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Human induced pluripotent stem cells (hiPSCs) are promising resources for producing various types of tissues in regenerative medicine; however, the improvement in a scalable culture system that can precisely control the cellular status of hiPSCs is needed. Utilizing suspension culture without microcarriers or special materials allows for massive production, automation, cost-effectiveness, and safety assurance in industrialized regenerative medicine. Here, we found that hiPSCs cultured in suspension conditions with continuous agitation without microcarriers or extracellular matrix components were more prone to spontaneous differentiation than those cultured in conventional adherent conditions. Adding PKCβ and Wnt signaling pathway inhibitors in the suspension conditions suppressed the spontaneous differentiation of hiPSCs into ectoderm and mesendoderm, respectively. In these conditions, we successfully completed the culture processes of hiPSCs, including the generation of hiPSCs from peripheral blood mononuclear cells with the expansion of bulk population and single-cell sorted clones, long-term culture with robust self-renewal characteristics, single-cell cloning, direct cryopreservation from suspension culture and their successful recovery, and efficient mass production of a clinical-grade hiPSC line. Our results demonstrate that precise control of the cellular status in suspension culture conditions paves the way for their stable and automated clinical application.

Myelin basic protein (MBP) is a major component of the myelin sheaths of oligodendrocytes in the central nervous system and Schwann cells of the peripheral nervous system. Here we generated heterozygous fluorescent reporter of MBP gene in human induced pluripotent stem cells (hiPSCs). CRISPR/Cas9 genome editing technology was employed to knock in fused tdTomato fluorescent protein and EF1 alpha promoter-driven Bleomycin (Zeocin) resistance gene to the translational MBP C-terminal region. The resulting line, MBP-TEZ, showed tdTomato fluorescence upon oligodendrocyte differentiation. This reporter hiPSC line provides a precedential opportunity for monitoring human myelin formation and degeneration and purifying MBP-expressing cell lineages.

The aerodynamic characteristics of a conventional line pantograph in a crosswind were investigated by conducting large-eddy simulations (LESs). In a previous study, wind tunnel tests using a full-scale pantograph were performed at various attack angles of the crosswind to understand the lift force characteristics of a pantograph in crosswind conditions. These experiments revealed that the lift force increases significantly when the yaw angle is set to 56°; however, the mechanism of increment of the lift force has not been clarified. Therefore, the flow fields around the pantograph head were investigated. It was found that there are two main mechanisms: one is a stationary large-scale vortex generated on the upper surface of the pantograph head, and the other is a pressure increment on the lower surface of the pantograph head. In addition, LESs were conducted using modified pantograph head shapes to investigate methods for reducing the lift force of the pantograph head using the mechanisms above. Two changes to the pantograph head shape were considered: a diagonal cut on the lower surface of the pantograph head and opening sections on the upper and lower surfaces of the pantograph head. The former shape generates a negative pressure area under the pantograph head, while the latter attenuates the large-scale vortex on the upper surface of the pantograph head. Thus, they achieve lift reduction by different mechanisms and can be used together. The lift reduction rate reaches approximately 60% when both methods are used together, indicating that this is an effective lift force reduction method.

Abstract Background CRISPR-Cas9 gene-editing technology has facilitated the generation of knockout mice, providing an alternative to cumbersome and time-consuming traditional embryonic stem cell-based methods. An earlier study reported up to 16% efficiency in generating conditional knockout (cKO or floxed) alleles by microinjection of 2 single guide RNAs (sgRNA) and 2 single-stranded oligonucleotides as donors (referred herein as “two-donor floxing” method). Results We re-evaluate the two-donor method from a consortium of 20 laboratories across the world. The dataset constitutes 56 genetic loci, 17,887 zygotes, and 1718 live-born mice, of which only 15 (0.87%) mice contain cKO alleles. We subject the dataset to statistical analyses and a machine learning algorithm, which reveals that none of the factors analyzed was predictive for the success of this method. We test some of the newer methods that use one-donor DNA on 18 loci for which the two-donor approach failed to produce cKO alleles. We find that the one-donor methods are 10- to 20-fold more efficient than the two-donor approach. Conclusion We propose that the two-donor method lacks efficiency because it relies on two simultaneous recombination events in cis, an outcome that is dwarfed by pervasive accompanying undesired editing events. The methods that use one-donor DNA are fairly efficient as they rely on only one recombination event, and the probability of correct insertion of the donor cassette without unanticipated mutational events is much higher. Therefore, one-donor methods offer higher efficiencies for the routine generation of cKO animal models.

ISL1 encodes a member of the LIM/homeodomain family of transcription factors. This encoded protein plays central roles in the development of motor neuron, pancreas, and secondary heart field. Here we generated heterozygous fluorescent reporters of the ISL1 gene in human induced pluripotent stem cells (hiPSCs). CRISPR/Cas9 genome editing technology was employed to knock-in 2A-tdTomato and EF1 alpha promoter-driven Bleomycin resistance gene to the translational ISL1 C-terminal region. The resulting ISL1-TEZ lines showed tdTomato fluorescence upon motor neuron differentiation. These reporter iPSC lines provide opportunity for monitoring and purifying these related cell lineages.

In this study, a large-eddy simulation (LES) is conducted to investigate meandering airflows generated throughout the underbody of a real-shaped train model in open air. In our previous study, this was shown by an LES of airflows around a simplified train model. The shape of the underbody affects the flow. However, thus far, no systematic analyses that consider bogies have been conducted. Our experimental results qualitatively reproduced the power spectra of lateral velocity fluctuations in the underbody obtained using a 1:8.4-scale train model on a moving belt in a wind tunnel. Results on the effects of various bogie shapes suggested that the meandering flow was primarily affected by the time-averaged profile of streamwise velocity. Thus, the effects of the bogie shape on airflow are considerable. Comparing bogie shapes with and without a side cover, that without a side cover generated meandering airflow with a slightly smaller peak frequency than that with a side cover, and a completely smooth bogie generated no meandering airflows. It was confirmed that this LES was useful as a technical tool to develop measures to reduce meandering airflows around high-speed trains.

To clarify the mechanisms underlying airflow-induced vibrations of high-speed trains running through tunnels, large-eddy simulation of a large-scale flow structure around a simplified 6-car train model was conducted. Since actual trains run on one of the double track lines, the position of the train model was made to deviate from the tunnel center and hence the gap between one of the sides of the train and the tunnel wall is narrower than that of the other side. A train running in the open air was also calculated for comparison. The results of this study shed light on the generation mechanism of the pressure fluctuations acting on the side of high-speed trains as follows. Firstly, in the open air, the air velocity in the space between the underbody and the ground gradually decreases from the head toward the tail of the train. Thus, the air velocity is slower than that on both sides of the train, which generates shear flows near the bottom edges of both sides of the train. The shear flows cause large Karman vortex-like vortices (staggered Karman vortex street), which in turn lead to a meandering airflow beneath the underbody of the train. Secondly, in the tunnel, the air velocity not only in the gap between the underbody and the ground but also in the narrower gap between the side of the train and the tunnel wall gradually decreases from the head toward the tail of the train. In the same mechanism as the open air, a meandering airflow is generated throughout the side and underbody of the train and causes pressure fluctuations along the side of the train. Finally, it is demonstrated that the wavelength of pressure fluctuations along the side of the actual train can be estimated from the present LES results.

The recombinant adenoviral gene expression system is a powerful tool for gene delivery. However, it is difficult to obtain high titers of infectious virus, principally due to the toxicity of the expressed gene which affects on virus replication in the host HEK293 cells. To avoid these problems, we generated a Cre-loxP-regulated fluorescent universal vector (termed pAxCALRL). This vector produces recombinant adenoviruses that express the red fluorescent protein (RFP) instead of the inserted gene during proliferation, which limits toxicity and can be used to monitor viral replication. Expression of the gene of interest is induced by co-infection with an adenovirus that expresses Cre-recombinase (AxCANCre). Recombinant adenovirus produced by this system that express Hnf4α and Foxa2 were used to reprogram mouse embryo fibroblast (MEF) into induced-hepatocyte-like cells (iHep) following several rounds of infection, demonstrating the efficacy of this new system.

Under the existing method based on a detailed equation for evaluating the wind speed that can cause a vehicle to overturn, it is assumed that the crosswind blows the vehicle at the same wind speed uniformly over the entire length of the vehicle body along the track. However, taking into consideration the actual wind state, there is a wind speed distribution over the length of the vehicle body and there is a high possibility that the crosswind does not blow at the same wind speed over the entire length of the vehicle body. Therefore, if the wind speed distribution is considered, it becomes possible to evaluate the critical wind speed in more detail. In previous research, an evaluation formula of aerodynamic force in consideration of the wind speed distribution has been proposed with the natural wind direction assumed to be 90 degrees. On the other hand, the calculation of the wind speed that can cause a vehicle to overturn requires extending the evaluation formula so as to be applicable to the other natural wind directions. In this study we proposed, extending the existing evaluation formula, a method of calculating the critical wind speed in consideration of the spatial fluctuation of wind speed. We also evaluated the critical wind speed for the combination of the vehicle and the track constructions with the proposed method. In addition, we also evaluated the critical wind speed in accordance with changes in the height of the center of the car body from the ground and the roughness length. As a result of the evaluation, we confirmed that the critical wind speed increases by up to 1.4m/s, provided that the vehicles and railway structures are those assumed in this study.

A pantograph mounted on a high-speed train roof is exposed to high-speed air flow while traveling, and lift force is generated in the pantograph. To ensure high current collecting performance of the pantograph, it is important to appropriately adjust the lift force generated in the pantograph. The flow velocity along the roof of a train has a significant influence on the lift force. In this study, we proposed a simple calculation method that enabled us to easily calculate the flow on the train roof in a tunnel using a personal computer. To validate the effectiveness of the proposed method, model experiments were carried out and calculation and experimental results were compared. Furthermore, the influence of hydraulic friction coefficients on train roof flow velocity was examined using the proposed method, which was not time consuming.

In this study, the effect of glyceraldehyde 3-phosphate dehydrogenase (G3PD) on the molecular state of porcine myofibrils was investigated by observing the structural changes in myosin and actin in myofibrils using phase-contrast and fluorescence microscopy. Though the myofibril gel strength was not influenced by G3PD at a G3PD to myoflbril weight ratio (G/M ratio) of 1/20, the gel strength significantly increased at a G/M ratio ≥ 1/10. SDS-PAGE analysis demonstrated that myosin heavy chain band intensity increased in the myoflbril soluble fraction by adding G3PD, suggesting G3PD facilitated the solubilization of myosin and actin. Phase-contrast microscopy also showed increased myoflbril solubilization with increasing G3PD. Fluorescence microscopy revealed that G3PD colocalized with actin segments. Myosin segments also colocalized with actin segments in G3PD-treated myofibrils, suggesting myosin bound to actin. The addition of G3PD to myofibrils increased the Mg2+- and Mg2+-EGTA-ATPase activities, suggesting G3PD would not change the conformation of myofibrils. [ABSTRACT FROM AUTHOR]