Protocol for gene knockdown using siRNA in primary cultured neonatal murine microglia.

In this protocol, we describe the small interfering RNA (siRNA)-mediated gene knockdown in primary mouse microglia, providing an approach to investigate functions such as phagocytosis and chemotaxis. The approach includes siRNA design, establishment of mixed glial cultures, microglia isolation, and siRNA transfection. Validation of knockdown efficacy employs quantitative immunoblot analysis. This technique empowers the investigation of specific molecular and cellular functions within the intricate microenvironment of the brain, comprising diverse cell types. For complete details on the use and execution of this protocol, please refer to Iguchi et al. (2023).1.

Photocatalytic Activity of Ga2O3 Supported on Al2O3 for Water Splitting and CO2 Reduction.

We have examined the photocatalytic activity of Ga2O3 supported on Al2O3 (Ga2O3/Al2O3 catalyst) without a noble metal cocatalyst for water splitting and reduction of CO2 with water under UV light irradiation by changing the loading amount of Ga2O3. All prepared Ga2O3/Al2O3 catalysts show photocatalytic activities for both water splitting and CO2 reduction, and their activities are significantly improved compared to those of nonsupported Ga2O3 and Al2O3. The water splitting is dominated for Ga2O3/Al2O3 with less than 1.0 vol % of Ga2O3 loaded, whereas the CO2 reduction, for higher Ga2O3-loaded samples (2.6, 4.2 vol %). Crystalline structure characterizations of Ga2O3/Al2O3 catalysts indicate that active sites for both reactions are different. The water splitting proceeds on nanometer-sized Ga2O3 rods dispersed on an Al2O3 support consisting of a little distorted α-Ga2O3 phase. On the other hand, the CO2 reduction proceeds on sub-micrometer-sized Ga2O3 particles consisting of mixed phases of α-Ga2O3 and γ-Ga2O3 or with appearance of boundaries between the α and γ phases, which plays a critical role. Al2O3 used as the support of the Ga2O3 particles does not seem to play an important role in the photocatalytic CO2 reduction.

Realization of red shift of absorption spectra using optical near-field effect.

In many applications such as CO2 reduction and water splitting, high-energy photons in the ultraviolet region are required to complete the chemical reactions. However, to realize sustainable development, the photon energies utilized must be lower than the absorption edge of the materials including the metal complex for CO2 reduction, the electrodes for water splitting, because of the huge amount of lower energy than the visible region received from the sun. In the previous works, we had demonstrated that optical near-fields (ONFs) could realize chemical reactions, by utilizing photon energies much lower than the absorption edge because of the spatial non-uniformity of the electric field. In this paper, we demonstrate that an ONF can realize the red shift of the absorption spectra of the metal-complex material for photocatalytic reduction. By attaching the metal complex to ZnO nano-crystalline aggregates with nano-scale protrusions, the absorption spectra by using diffuse reflection of the metal complex can be shifted to a longer wavelength by 10.6 nm. The results of computational studies based on a first-principles computational program including the ONF effect provide proof of the increase in the absorption of the metal complex at lower photon energies. Since the near-field assisted field increase improves the carrier excitation in the metal-complex materials, this effect may be universal and it could applicable to CO2 reduction using the other metal-complex materials, as well as to the other photo excitation process including water splitting.

Effect of liquid flow by pipetting during medium change on deformation of hiPSC aggregates.

INTRODUCTION: Maintaining the pluripotency and homogeneity of human induced pluripotent stem cells (hiPSCs) requires stable culture conditions with consistent medium change. In this study, we evaluated the performance of medium change by machine vs. medium change performed manually in terms of their impact on the aggregate shape of hiPSCs. METHODS: Aggregates of two hiPSC lines (1383D2 and Tic) were cultured, and the medium change was conducted either manually or with a machine. The populational homogeneity in aggregate shape was determined based on the projected aggregate area for size expansion as well as the circularity for spherical morphology. RESULTS: In the case of manually performed medium changes, the size of 1383D2 aggregates expanded homogeneously, maintaining its spherical morphology as culture duration increased, while spherical morphology was deformed in Tic aggregates, which had a heterogeneous population in terms of shape. In the case of medium change performed by a machine under a low flux of liquid flow, cultures of both aggregates showed homogeneous populations without deformation, although a high flux led to a heterogeneous population. The heterogeneous population observed in manually performed medium change was caused by the low stability of motion. In addition, time-lapse observation revealed that the Tic aggregates underwent tardive deformation with cellular protrusions from the aggregate surface after medium change with high flux. Histological analysis revealed a spatial heterogeneity of collagen type I inside 1383D2 aggregates, which had a shell structure with strong formation of collagen type I at the periphery of the aggregates, while Tic aggregates did not have a shell structure, suggesting that the shell structure prevented aggregate deformation. CONCLUSION: Medium change by a machine led to a homogeneous population of aggregate shapes. Liquid flow caused tardive deformation of aggregates, but the shell structure of collagen type I in aggregates maintained its spherical shape.

Comparison of growth kinetics between static and dynamic cultures of human induced pluripotent stem cells.

Understanding the fundamental mechanisms that govern the growth kinetics of human induced pluripotent stem cells (hiPSCs) contributes to culture design strategies to improve large-scale production. Two hiPSC lines (Tic and 253G1) were cultured under static and dynamic suspension conditions, and growth kinetics were compared during early (24-48 h), middle (48-72 h), and late (72-96 h) stages. In 2D static culture, similar growth profiles were observed for both hiPSC lines. However, there were significant differences in growth profile patterns and aggregate morphologies between hiPSC lines grown in 3D static and dynamic cultures. Based on immunostaining comparing the two hiPSC lines, surface distribution of collagen type I was observed in aggregates of the Tic line, but not in those of the 253G1 line. Compared to that in 3D static culture, the numbers of cells at 96 h were significantly decreased in 3D dynamic culture. The apparent specific growth rate (µapp) of the Tic line was maintained continuously throughout culture, whereas that of the 253G1 line decreased gradually with culture until the late phase, at which time this parameter was reduced to µapp = (0.85 ± 0.71) × 10-2 h-1. This indicates that during the growth of hiPSCs in 3D dynamic culture, cells were damaged by liquid flow, which disrupted the cell-synthesized extracellular matrix (ECM). These results demonstrate that cell-synthesized ECM is an important factor affecting cell growth and morphology, and that changes to the ECM within aggregates lead to reduced growth abilities in dynamic culture.

FLOWERING LOCUS T regulates stomatal opening.

Stomatal pores surrounded by a pair of guard cells in the plant epidermis control gas exchange for photosynthesis in response to light, CO(2), and phytohormone abscisic acid. Phototropins (phot1 and phot2) are plant blue-light receptor kinases and mediate stomatal opening via activation of the plasma membrane H(+)-ATPase. However, the signaling mechanism from phototropins to the H(+)-ATPase has yet to be determined. Here, we show that FLOWERING LOCUS T (FT) is expressed in guard cells and regulates stomatal opening. We isolated an scs (suppressor of closed-stomata phenotype in phot1 phot2) 1-1 mutant of Arabidopsis thaliana and showed that scs1-1 carries a novel null early flowering 3 (elf3) allele in a phot1 phot2 background. scs1-1 (elf3 phot1 phot2 triple mutant) had an open-stomata phenotype with high H(+)-ATPase activity and showed increased levels of FT mRNA in guard cells. Transgenic plants overexpressing FT in guard cells showed open stomata, whereas a loss-of-function FT allele, ft-1, exhibited closed stomata and failed to activate the H(+)-ATPase in response to blue light. Our results define a new cell-autonomous role for FT and demonstrate that the flowering time genes ELF3 and FT are involved in the regulation of H(+)-ATPase by blue light in guard cells.