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The dynamics of oxytocin and its site of action in the brain are poorly understood due to the lack of appropriate tools, despite the interest in the central action of oxytocin signaling. Here, we develop and apply an oxytocin analogue probe by conjugating it with an alkyne

The dopaminergic system is essential for the function of the brain in health and disease. Therefore, detailed studies focused on unraveling the mechanisms involved in dopaminergic signaling are required. However, the lack of probes that mimic dopamine in living tissues, owing to the neurotransmitter's small size, has hampered analysis of the dopaminergic system. The current study aimed to overcome this limitation by developing alkyne-tagged dopamine compounds (ATDAs) that have a minimally invasive and uniquely identifiable alkyne group as a tag. ATDAs were established as chemically and functionally similar to dopamine and readily detectable by methods such as specific click chemistry and Raman scattering. The ATDAs developed here were verified as analogue probes that mimic dopamine in neurons and brain tissues, allowing the detailed characterization of dopamine dynamics. Therefore, ATDAs can act as safe and versatile tools with wide applicability in detailed studies of the dopaminergic system. Furthermore, our results suggest that the alkyne-tagging approach can also be applied to other small-sized neurotransmitters to facilitate characterization of their dynamics in the brain.

Cells within the brain are highly organized and coordinate complex processes with each other. The ability to simultaneously visualize the organization and interactions of cells and molecules within brain tissue remains an important issue to understand the brain comprehensively. Stimulated Raman scattering (SRS) and fluorescence, two powerful imaging modalities, can provide complementary molecular contrasts within cells and tissue samples. Here, we present a high-speed super-multiplex imaging platform that combines SRS microscopy with confocal fluorescence microscopy to perform rapid 7-color brain imaging. We show simultaneous imaging of cellular components within the brain such as astrocytes, axons, and blood vessels while also showing organelles such as the nucleus and actin cytoskeleton. Also, we demonstrate the ability to take depth-resolved images that elucidate the three-dimensional organization of diverse components within brain tissue. This platform can be adapted to explore various processes within brain tissue that can reveal critical information about the brain and how it is affected by diseases, which leads toward a deeper understanding of disease progression and potentially the development of therapeutic options for brain diseases.

Summary Observing multiple molecular species simultaneously with high spatiotemporal resolution is crucial for comprehensive understanding of complex, dynamic, and heterogeneous biological systems. The recently reported super-multiplex optical imaging breaks the “color barrier” of fluorescence to achieve multiplexing number over six in living systems, while its temporal resolution is limited to several minutes mainly by slow color tuning. Herein, we report integrated stimulated Raman and fluorescence microscopy with simultaneous multimodal color tunability at high speed, enabling super-multiplex imaging covering diverse molecular contrasts with temporal resolution of seconds. We highlight this technique by demonstrating super-multiplex time-lapse imaging and image-based cytometry of live cells to investigate the dynamics and cellular heterogeneity of eight intracellular components simultaneously. Our technique provides a powerful tool to elucidate spatiotemporal organization and interactions in biological systems., Graphical Abstract, Highlights • Integrated SRS and fluorescence microscopy with fast tunability has been developed • Eight-color live-cell imaging can be conducted with a temporal resolution of seconds • Super-multiplex time-lapse imaging reveals complex organelle interactions • Super-multiplex image-based cytometry accesses high-dimensional heterogeneity, Optics; Cell biology; Biological sciences research methodologies

Hereditary deafness affects approximately 1 in 2,000 children. Mutations in the gene encoding the cochlear gap junction protein connexin 26 (CX26) cause prelingual, nonsyndromic deafness and are responsible for as many as 50% of hereditary deafness cases in certain populations. Connexin-associated deafness is thought to be the result of defective development of auditory sensory epithelium due to connexion dysfunction. Surprisingly, CX26 deficiency is not compensated for by the closely related connexin CX30, which is abundantly expressed in the same cochlear cells. Here, using two mouse models of CX26-associated deafness, we demonstrate that disruption of the CX26-dependent gap junction plaque (GJP) is the earliest observable change during embryonic development of mice with connexin-associated deafness. Loss of CX26 resulted in a drastic reduction in the GJP area and protein level and was associated with excessive endocytosis with increased expression of caveolin 1 and caveolin 2. Furthermore, expression of deafness-associated CX26 and CX30 in cell culture resulted in visible disruption of GJPs and loss of function. Our results demonstrate that deafness-associated mutations in CX26 induce the macromolecular degradation of large gap junction complexes accompanied by an increase in caveolar structures.

Background: Induced pluripotent stem (iPS) cells aremultipotentstem cellsthat can be producedfrom adult cells such as fibroblastsby the induction ofartificial reprogramming. Since such cells can be differentiated to endoderm, mesodermand ectoderm asembryonic stem (ES) cells, theirpotential utility for the regenerative therapy of various organs is expected. The inner ear is composed of various types of functional cells (hair cells, supporting cells, spiral ganglion, cochlear fibrocytes, striavascularis, etc.), and each cell type. Although the degeneration of these cells leads directly to severe hearing loss, there are few reports concerning thedifferentiation of iPS cells to various types of inner ear cells for regenerative therapy. Results: In this study, we co-cultured iPS-derived cells with three different regions (spiral ganglion, the organ of Corti with spiral limbus, lateral wall) of the cochlear tissue and tod attempted to induce their differentiation into various types of inner ear cells. The number of positive colonies (Nanogpositive colony) with reporter GFP controlled by Nanogpromotor was counted to assess the undifferentiated level of the cells. In the co-cultured cells with spiral ganglion, many Nanog-positive colonies were observed, and many cells with neurite-like protrusions were observed among the colonies. In the co-culture with the organ of Cortiand spiral limbus (OC/SL), neuron-like cells were observed on the cell layer with tight junction-like adhesions. In the co-culture with the cochlear lateral wall, many cell layers with tight junction-like adhesions were observed, while undifferentiated Nanogpositive colonies were not observed. Conclusion: In this study, we demonstrated that differentiation of iPS cells to cochlear cells was regulated depending on the part of the co-cultured organs. Co-culture with cochlea tissue and the induced pluripotent stem cells may enable regenerative therapy of various types of inner ear cells.

Background The greater epithelial ridge (GER) is a developmental structure in the maturation of the organ of Corti. Situated near the inner hair cells of neonatal mice, the GER undergoes a wave of apoptosis after postnatal day 8 (P8). We evaluated the GER from P8 to P12 in transgenic mice that carry the R75W + mutation, a dominant-negative mutation of human gap junction protein, beta 2, 26 kDa (GJB2) (also known as connexin 26 or CX26). Cx26 facilitate intercellular communication within the mammalian auditory organ. Results In both non-transgenic (non-Tg) and R75W + mice, some GER cells exhibited apoptotic characteristics at P8. In the GER of non-Tg mice, both the total number of cells and the number of apoptotic cells decreased from P8 to P12. In contrast, apoptotic cells were still clearly evident in the GER of R75W + mice at P12. In R75W + mice, therefore, apoptosis in the GER persisted until a later stage of cochlear development. In addition, the GER of R75W + mice exhibited morphological signs of retention, which may have resulted from diminished levels of apoptosis and/or promotion of cell proliferation during embryogenesis and early postnatal stages of development. Conclusions Here we demonstrate that Cx26 dysfunction is associated with delayed apoptosis of GER cells and GER retention. This is the first demonstration that Cx26 may regulate cell proliferation and apoptosis during development of the cochlea.

優良系統豚を利用した良食味豚肉の開発に関する研究の中で, 豚肉の食味性について検討を試み, 官能検査に係わる以下の結果を得た。(1) 官能検査における豚肉の調理条件は, 厚さ2.5mmで一定とし, 試料の2倍量の1.5%食塩水を用いた時, 肉本来の味の判定がしやすいことが分かった。(2) 冷凍肉は解凍によって, 水分6～8%, タンパク質3～4%が失われ, 官能検査の「多汁性」に影響を与えた。(3) 肉は加熱調理により, 重量, 表面積は減少し, 厚さは増加したが, その程度はいずれも冷蔵に比べ冷凍の方が大となった。(4) 肉の硬さは加熱により3～4倍に増加したが, 冷凍は冷蔵より厚くなったにもかかわらず, 官能検査では「軟らかい」と評価された。(5) 肉の冷凍保存は官能検査に多くの影響を及ぼした。冷凍に際しては, 凍結, 包装, 保存温度など, 各条件を統一する必要がある。

The survival of yeast in frozen dough in several stages in the baking process up to 10 weeks of frozen storage and the rheological properties of thawed dough were determined to clarify the causes of bread deterioration during long storage. Torulaspora delbrueckii was used as a freeze tolerant yeast and Saccharomyces cereuisiae as a freeze sensitive yeast. The survival yeast counts were constant in several steps of the breadmaking process before baking in non-frozen and frozen dough with both types of yeasts. The frozen dough using T. delbrueckii retained constant survival yeast counts for 10 weeks, but there was a decrease in the fermentative ability per yeast cell during longer storage. With white bread dough using T. delbrueckii, the fermentative ability gradually decreased, and with sweet bread dough, it decreased quickly from 4 weeks on. The survival counts of yeast on S. cereuisiae were 30% for white bread dough after 1 week storage and 70% for sweet bread dough. The changes in the rheological properties started immediately after freezing and the adhesiveness of thawed dough increased with the length of the storage period in every case regardless of the survival ratio of yeast cells. The long storage of frozen dough with T. delbrueckii has a negative effect on the quality of bread and appliable storage period is under 3 weeks.

Brn4, which encodes a POU transcription factor, is the gene responsible for DFN3, an X chromosome-linked, non-syndromic type of hearing loss. Brn4-deficient mice have a low endocochlear potential (EP), hearing loss, and ultrastructural alterations in spiral ligament fibrocytes, however the molecular pathology through which Brn4 deficiency causes low EP is still unclear. Mutations in the Gjb2 and Gjb6 genes encoding the gap junction proteins connexin26 (Cx26) and connexin30 (Cx30) genes, respectively, which encode gap junction proteins and are expressed in cochlear fibrocytes and non-sensory epithelial cells (i.e., cochlear supporting cells) to maintain the proper EP, are responsible for hereditary sensorineural deafness. It has been hypothesized that the gap junction in the cochlea provides an intercellular passage by which K+ is transported to maintain the EP at the high level necessary for sensory hair cell excitation. Here we analyzed the formation of gap junction plaques in cochlear supporting cells of Brn4-deficient mice at different stages by confocal microscopy and three-dimensional graphic reconstructions. Gap junctions from control mice, which are composed mainly of Cx26 and Cx30, formed linear plaques along the cell-cell junction sites with adjacent cells. These plaques formed pentagonal or hexagonal outlines of the normal inner sulcus cells and border cells. Gap junction plaques in Brn4-deficient mice did not, however, show the normal linear structure but instead formed small spots around the cell-cell junction sites. Gap junction lengths were significantly shorter, and the level of Cx26 and Cx30 was significantly reduced in Brn4-deficient mice compared with littermate controls. Thus the Brn4 mutation affected the assembly and localization of gap junction proteins at the cell borders of cochlear supporting cells, suggesting that Brn4 substantially contributes to cochlear gap junction properties to maintain the proper EP in cochleae, similar to connexin-related deafness.

Plasmodia were fractionated to isolate a cell membrane rich fraction by sucrose density-gradient centrifugation. The fractions were identified by electron microscopic observation, PTA-chromic acid staining and assays of marker enzymes, applying the methods for cell membranes of higher plants. The cell membranes were recovered on the density of 1.13 g·cm−3.

1. 1. The cranial cartilage of the squid Todarodes pacificus , which resembles vertebrate hyaline cartilage in its microscopic appearance, was solubilized by limited proteolysis with pepsin. 2. 2. Characterization of the solubilized collagen suggests that the squid cartilage contains primarily Type I collagen which is rich in hydroxylysine-linked carbohydrate, similar to the cartilage-type collagen of vertebrates. 3. 3. Tissue-specific differences in proline hydroxylation and thermal stability of squid Type I collagen were observed, comparing the collagens of cranial cartilage and mantle skin tissues.

The nutritive value of various carbohydrates and the supplementary effect of inositol were investigated.Male Wister young rats were maintained by basic diet containing 65.5% sucrose, 22.0% casein, 9.0% oil, 2.5% salts and 1.0% vitamins. Then 3% sucrose in the basal diet were substituted by other carbohydrates; mono or oligo saccharide group (glucose, lactose, xylose), poly saccharide group (dextrin, α-cornstarch, β-cornstarch) and non calorie poly saccharide group (pectin, agar, cellulose).Each experimental group was divided in to two groups with or without inositol.The experimental results are:1) Glucose, β-cornstarch and cellulose effected increasing the weight of the rats but experiments that used the other carbohydrates proved to have no effects.2) Addition of inositol to mono-, di saccharide and poly saccharide well effected increasing the weight of the rats. But addition of inositol gave no effects to non calorie poly saccharide group.3) Diet efficiency, protein efficiency, and calorie efficiency raised on glucose, β-cornstarch and lactose with inositol.

The comparative studies on the morphological and physiological characteristics of the cultures of blastmycin and antimycin A sources indicated that most of the strains may be related to each other. This idea was confirmed by the marked cross precipitin reactions of these organisms. They may be classified into three groups A, B-1 and B-2. The antimycin A-producing organism belongs to the former group and the blastmycin ones to the latter two groups. The absorption tests and OUCHTERLONY's gel diffusion methods suggested that three groups of the streptomyces may be serologically independant. The antigenic structure of the strain No. 455-D1 appear to be more complicated than those of the strains No. 2A-720 and No. 709-A1. They contain the specific antigen(s) and common antigen(s). A strain of antimycin A sources was observed to belong to the species Streptomyces griseus. It showed no cross precipitin reactions with the other organisms.