Rapid Fabricating Technique for Multi-Layered Human Hepatic Cell Sheets by Forceful Contraction of the Fibroblast Monolayer

Cell sheet engineering is attracting attention from investigators in various fields, from basic research scientists to clinicians focused on regenerative medicine. However, hepatocytes have a limited proliferation potential in vitro, and it generally takes a several days to form a sheet morphology and multi-layered sheets. We herein report our rapid and efficient technique for generating multi-layered human hepatic cell (HepaRG® cell) sheets using pre-cultured fibroblast monolayers derived from human skin (TIG-118 cells) as a feeder layer on a temperature-responsive culture dish. Multi-layered TIG-118/HepaRG cell sheets with a thick morphology were harvested on day 4 of culturing HepaRG cells by forceful contraction of the TIG-118 cells, and the resulting sheet could be easily handled. In addition, the human albumin and alpha 1-antitrypsin synthesis activities of TIG-118/HepaRG cells were approximately 1.2 and 1.3 times higher than those of HepaRG cells, respectively. Therefore, this technique is considered to be a promising modality for rapidly fabricating multi-layered human hepatocyte sheets from cells with limited proliferation potential, and the engineered cell sheet could be used for cell transplantation with highly specific functions.     

Environmental DNA as a ‘Snapshot’ of Fish Distribution: A Case Study of Japanese Jack Mackerel in Maizuru Bay,Sea of Japan

Recent studies in streams and ponds have demonstrated that the distribution and biomass of aquatic organisms can be estimated by detection and quantification of environmental DNA (eDNA). In more open systems such as seas, it is not evident whether eDNA can represent the distribution and biomass of aquatic organisms because various environmental factors (e.g., water flow) are expected to affect eDNA distribution and concentration. To test the relationships between the distribution of fish and eDNA, we conducted a grid survey in Maizuru Bay, Sea of Japan, and sampled surface and bottom waters while monitoring biomass of the Japanese jack mackerel (Trachurus japonicus) using echo sounder technology. A linear model showed a high R² value (0.665) without outlier data points, and the association between estimated eDNA concentrations from the surface water samples and echo intensity was significantly positive, suggesting that the estimated spatial variation in eDNA concentration can reflect the local biomass of the jack mackerel. We also found that a best-fit model included echo intensity obtained within 10–150 m from water sampling sites, indicating that the estimated eDNA concentration most likely reflects fish biomass within 150 m in the bay. Although eDNA from a wholesale fish market partially affected eDNA concentration, we conclude that eDNA generally provides a ‘snapshot’ of fish distribution and biomass in a large area. Further studies in which dynamics of eDNA under field conditions (e.g., patterns of release, degradation, and diffusion of eDNA) are taken into account will provide a better estimate of fish distribution and biomass based on eDNA.     

The role of floral structure and biotic factors in determining the occurrence of florivorous thrips in a dystilous shrub

Elucidating the factors determining the occurrence of florivorous organisms is an essential step for comprehending arthropod–plant interactions, especially when considering florivores that use flowers/inflorescences as microhabitats. In this study, we characterize the interaction between florivorous thrips (Thysanoptera) and Palicourea rigida (Rubiaceae), a distylous hummingbird-pollinated shrub. We investigated the relative role of different factors in determining thrips occurrence in the flower and inflorescence microhabitats. Furthermore, we experimentally examined the protective role of corolla influencing thrips exploration of floral buds. Frankliniella musaeperda (Thripidae) was the only species recorded on P. rigida, feeding on floral tissue, pollen and nectar. Thrips occurrence was not related to distyly, but rather to floral stage. Open flowers presented the highest abundance of thrips, followed by senescent flowers and then buds. The experimental opening of buds translated in increased thrips occurrence, indicating that F. musaeperda manage to explore the microhabitat offered by the floral chamber, as long as there is an opening in the corolla. In inflorescences, thrips abundance was negatively related to the number of ants visiting extrafloral nectaries. We found that the marked difference between floral morphs of distylous plants is not necessarily reflected in the abundance of florivores. Thrips seek for floral cavities, preferentially those with fresh tissue, which may confer nutrient-rich food and protection. Buds also provide this; however, the enclosed petals are an effective barrier against F. musaeperda entrance. At inflorescence scale, presence of mutualistic ants in high numbers can drive away these flower-feeding insects. Despite the abundance of thrips in the flowers, there was no evidence of any functional relationship, either of pollination for flowers or of breeding for insects. We demonstrate here that in the flower/inflorescence microhabitat, structural and biotic factors play a key role in the exploitation and occupation by insect florivores.     

Reappraisal of VAChT‐Cre: Preference in slow motor neurons innervating type I or IIa muscle fibers

VAChT‐Cre.Fast and VAChT‐Cre.Slow mice selectively express Cre recombinase in approximately one half of postnatal somatic motor neurons. The mouse lines have been used in various studies with selective genetic modifications in adult motor neurons. In the present study, we crossed VAChT‐Cre lines with a reporter line, CAG‐Syp/tdTomato, in which synaptophysin‐tdTomato fusion proteins are efficiently sorted to axon terminals, making it possible to label both cell bodies and axon terminals of motor neurons. In the mice, Syp/tdTomato fluorescence preferentially co‐localized with osteopontin, a recently discovered motor neuron marker for slow‐twitch fatigue‐resistant (S) and fast‐twitch fatigue‐resistant (FR) types. The fluorescence did not preferentially co‐localize with matrix metalloproteinase‐9, a marker for fast‐twitch fatigable (FF) motor neurons. In the neuromuscular junctions, Syp/tdTomato fluorescence was detected mainly in motor nerve terminals that innervate type I or IIa muscle fibers. These results suggest that the VAChT‐Cre lines are Cre‐drivers that have selectivity in S and FR motor neurons. In order to avoid confusion, we have changed the mouse line names from VAChT‐Cre.Fast and VAChT‐Cre.Slow to VAChT‐Cre.Early and VAChT‐Cre.Late, respectively. The mouse lines will be useful tools to study slow‐type motor neurons, in relation to physiology and pathology.     

Single‐Walled Carbon Nanotubes in Emerging Solar Cells: Synthesis and Electrode Applications

Emerging solar cells, namely, organic solar cells and perovskite solar cells, are the thin‐film photovoltaics that have light to electricity conversion efficiencies close to that of silicon solar cells while possessing advantages in having additional functionalities, facile‐processability, and low fabrication cost. To maximize these advantages, the electrode components must be replaced by materials that are more flexible and cost‐effective. Researchers around the globe have been looking for the new electrodes that meet these requirements. Among many candidates, single‐walled carbon nanotubes have demonstrated their feasibility as the new alternative to conventional electrodes, such as indium tin oxide and metals. This review discusses various growth methods of single‐walled carbon nanotubes and their electrode applications in thin‐film photovoltaics.     

Competition for Mitogens Regulates Spermatogenic Stem Cell Homeostasis in an Open Niche

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Acoustic emissions of Sorex unguiculatus (Mammalia: Soricidae): Assessing the echo‐based orientation hypothesis

Shrew species have been proposed to utilize an echo‐based orientation system to obtain additional acoustic information while surveying their environments. This system has been supported by changes in vocal emission rates when shrews encounter different habitats of varying complexity, although detailed acoustic features in this system have not been reported. In this study, behavioral experiments were conducted using the long‐clawed shrew (Sorex unguiculatus) to assess this orientation system. Three experimental conditions were set, two of which contained obstacles. Short‐click, noisy, and different types of tonal calls in the audible‐to‐ultrasonic frequency range were recorded under all experimental conditions. The results indicated that shrews emit calls more frequently when they are facing obstacles or exploring the experimental environment. Shrews emitted clicks and several different types of tonal calls while exploring, and modified the use of different types of calls for varying behavior. Furthermore, shrews modified the dominant frequency and duration of squeak calls for different types of obstacles, that is, plants and acrylic barriers. The vocalizations emitted at short inter‐pulse intervals could not be observed when shrews approached these obstacles. These results are consistent with the echo‐based orientation hypothesis according to which shrews use a simple echo‐orientation system to obtain information from their surrounding environments, although further studies are needed to confirm this hypothesis.     

Investigation of the dimerization of proteins from the epidermal growth factor receptor family by single wavelength fluorescence cross-correlation spectroscopy

Single wavelength fluorescence cross-correlation spectroscopy (SW-FCCS), introduced to study biomolecular interactions, has recently been reported to monitor enzyme activity by using a newly developed fluorescent protein variant together with cyan fluorescent protein. Here, for the first time to our knowledge, SW-FCCS is applied to detect interactions between membrane receptors in vivo by using the widely used enhanced green fluorescent protein and monomeric red fluorescent protein. The biological system studied here is the epidermal growth factor/ErbB receptor family, which plays pivotal roles in the development of organisms ranging from worms to humans. It is widely thought that a ligand binds to the monomeric form of the receptor and induces its dimeric form for activation. By using SW-FCCS and F?rster resonance energy transfer, we show that the epidermal growth factor receptor and ErbB2 have preformed homo- and heterodimeric structures on the cell surface and quantitation of dimer fractions is performed by SW-FCCS. These receptors are major targets of anti-cancer drug development, and the receptors' homo- and heterodimeric structures are relevant for such developments.     

Genetic Diversity of the Cryptococcus gattii Species Complex in Mato Grosso State,Brazil

Mycopathologia - Cryptococcosis is caused by fungi of the genus Cryptococcus. Owing to its importance, this study aimed to analyze the genetic diversity of C. gattii isolates from animals, humans,...