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Abstract Background Resident memory T (TRM) cells and immunosuppressive Foxp3‐expressing regulatory TRM (regTRM) cells are present in healthy, non‐inflamed and inflamed human skin. Both types of cells are found in both the epidermis and dermis with the dermis being much thicker than the epidermis. However, it is unclear if TRM and regTRM cells differ between reticular dermal layers in terms of number, function or characteristics. Methods This study examined numerical, phenotypic and functional differences in TRM and regTRM cells between the upper and lower reticular dermis in healthy, non‐inflamed human skin using flow cytometry. Results The phenotype and cytokine expressions of both types of cells were similar between reticular dermal layers. However, the cell count of both TRM and regTRM cells was significantly higher in the upper reticular dermis than in the lower reticular dermis. Conclusions TRM and regTRM cells are phenotypically and functionally identical between reticular dermal layers. However, both types of cells are more abundant in the upper reticular dermis.

Incorporation of bovine serum-derived albumin formulation (AlbuMAX) into a basic culture medium, MEMα, enables the completion of in vitro spermatogenesis through testicular tissue culture in mice. However, this medium was not effective in other animals. Therefore, we sought an alternative approach for in vitro spermatogenesis using a synthetic medium without AlbuMAX and aimed to identify its essential components. In addition to factors known to be important for spermatogenesis, such as retinoic acid and reproductive hormones, we found that antioxidants (vitamin E, vitamin C, and glutathione) and lysophospholipids are vital for in vitro spermatogenesis. Moreover, based on our experience with microfluidic devices (MFD), we developed an alternative approach, the PDMS-ceiling method (PC method), which involves simply covering the tissue with a flat chip made of PDMS, a silicone resin material used in MFD. The PC method, while straightforward, integrates the advantages of MFD, enabling improved and uniform oxygen and nutrient supply via tissue flattening. Furthermore, our studies underscored the significance of lowering the oxygen concentration to 10–15%. Using an integrated cultivation method based on these findings, we successfully achieved in vitro spermatogenesis in rats, which has been a long-standing challenge. Further improvements in culture conditions would pave the way for spermatogenesis completion in diverse animal species.

Abstract Dinitrogen (N2) fixation provides the major source of reactive nitrogen in the open ocean, sustaining biological productivity. The Indian Ocean (IO) covers 22% of the ocean surface, while it only represents 1% of the global diazotroph database. Hence, constraining the sources of nitrogen in the IO is crucial. Here, we compile three decades of N2 fixation and diazotroph DNA data in the IO. Our analysis reveals basin‐scale yearly rates between ~ 7 and 13 Tg N yr−1. These rates are in the range of previous modeling‐based estimates but may represent a lower bound estimate due to the lack of data in this basin. Diazotroph variability among sub‐basins may suggest endemicity but needs to be taken with caution due to biased sampling toward certain seasons and uneven spatial coverage. We provide recommendations for a more accurate representation of the IO in the global nitrogen budget and our knowledge of diazotroph biogeography.

Abstract The classical organ culture method, in which tissue is placed at the gas‒liquid interphase, is effective at inducing mouse spermatogenesis. However, due to reginal variations in the supply of oxygen and nutrients within a tissue, the progress of spermatogenesis was observed only in limited areas of a tissue. In addition, haploid cell formation and its differentiation to spermatozoon, i.e. spermiogenesis, were infrequent and inefficient. Here, we show that the polydimethylsiloxane (PDMS)-chip ceiling (PC) method, which ensures a uniform supply of nutrients and oxygen throughout the tissue by pressing it into a thin, flat shape, can provide control over the culture space. We used this method to culture testis tissue from neonatal mice, aged 1 to 4 days, and found that modulating the culture space during the experiment by replacing one chip with another that had a higher ceiling effectively increased tissue growth. This adjustment also induced more efficient spermatogenesis, with the process of spermiogenesis being particularly promoted. Meiotic cells were observed from culture day 14 onward, and haploid cells were confirmed at the end of each experiment. This technique was also shown to be a sensitive assay for testicular toxicity. Culture-space control will be a critical regulation parameter for sophisticated tissue culture experiments.

Abstract An in vitro spermatogenesis method using mouse testicular tissue to produce fertile sperm was established more than a decade ago. Although this culture method has generally not been effective in other animal species, we recently succeeded in improving the culture condition to induce spermatogenesis of rats up to the round spermatid stage. In the present study, we introduced acrosin-EGFP transgenic rats in order to clearly monitor the production of haploid cells during spermatogenesis in vitro. In addition, a metabolomic analysis of the culture media during cultivation revealed the metabolic dynamics of the testis tissue. By modifying the culture media based on these results, we were able to induce rat spermatogenesis repeatedly up to haploid cell production, including the formation of elongating spermatids, which was confirmed histologically and immunohistochemically. Finally, we performed a microinsemination experiment with in vitro produced spermatids, which resulted in the production of healthy and fertile offspring. This is the first demonstration of the in vitro production of functional haploid cells that yielded offspring in animals other than mice. These results are expected to provide a basis for the development of an in vitro spermatogenesis system applicable to many other mammals.

After the Great East Japan earthquake, measures for beyond design basis events have become more important. For a component, mitigation of an influence of a failure on its safety performance is required in addition to prevention of the failure. These kinds of measures are reduced the risk of safety performance defined as multiplication of failure frequency and failure consequence regarding impact on safety performance. One of safety analyses regarding such measures is fragility analysis. However, the existing fragility is represented using failure probability, and does not take into account the failure consequence. In this paper, for the purpose of more rational fragility analysis, we propose a new concept of performance-based fragility incorporating failure consequence, which represents the risk of safety performance degradation. In addition, to present the effectiveness, the proposed fragility is quantified by applying to a piping system subjected to excessive earthquakes which is a key issue of beyond design basis event. A piping system subjected to excessive earthquake can fail by ratchet, fatigue, collapse, and break. These failure modes have different occurrence conditions and lead to different consequences about core cooling performance. After the analyses on occurrence probability and failure consequence on each failure modes, the proposed fragility is calculated as the multiplication of them.

Increased levels of lactate, an end-product of glycolysis, have been proposed as a potential surrogate marker for metabolic changes during neuronal excitation. These changes in lactate levels can result in decreased brain pH, which has been implicated in patients with various neuropsychiatric disorders. We previously demonstrated that such alterations are commonly observed in five mouse models of schizophrenia, bipolar disorder, and autism, suggesting a shared endophenotype among these disorders rather than mere artifacts due to medications or agonal state. However, there is still limited research on this phenomenon in animal models, leaving its generality across other disease animal models uncertain. Moreover, the association between changes in brain lactate levels and specific behavioral abnormalities remains unclear. To address these gaps, the International Brain pH Project Consortium investigated brain pH and lactate levels in 109 strains/conditions of 2294 animals with genetic and other experimental manipulations relevant to neuropsychiatric disorders. Systematic analysis revealed that decreased brain pH and increased lactate levels were common features observed in multiple models of depression, epilepsy, Alzheimer’s disease, and some additional schizophrenia models. While certain autism models also exhibited decreased pH and increased lactate levels, others showed the opposite pattern, potentially reflecting subpopulations within the autism spectrum. Furthermore, utilizing large-scale behavioral test battery, a multivariate cross-validated prediction analysis demonstrated that poor working memory performance was predominantly associated with increased brain lactate levels. Importantly, this association was confirmed in an independent cohort of animal models. Collectively, these findings suggest that altered brain pH and lactate levels, which could be attributed to dysregulated excitation/inhibition balance, may serve as transdiagnostic endophenotypes of debilitating neuropsychiatric disorders characterized by cognitive impairment, irrespective of their beneficial or detrimental nature.

Monitoring the movement of small animals is a fundamental aspect of ecological studies as well as spatially explicit conservation and management. However, this remains a challenging task especially in mountainous terrains. Although drone-based radiotelemetry (DRT) is employed to localize animals, its application in mountainous terrains is limited by the collision risks associated with undulating terrains as well as the obstruction of signals by dense vegetation and steep slopes. We addressed these challenges by generating fine-scale three-dimensional maps and moving vertically mounted directional antennas in a double grid pattern, scanning both in longitudinal and latitudinal grids. This new DRT system was helpful in localizing four adult Japanese toads (Bufo japonicus) living in hiding places typical of mountainous terrains. All toads were located within 1–60 days of being released. Transmitter signals were detected within two consecutive flights (three flights in one case). Instances of transmitter detection were significantly biased when the drone was facing either direction of the double-grid path, indicating that the double-grid pattern had reduced detection failure. The absolute localization error (n = 48) of 22.4 ± 21.0 m (44.8 ± 42% of the transmitter-to-receiver distance) was lower than that reported in a previous study conducted in a similar mountainous terrain.

Abstract Spontaneous activity during the early postnatal period is thought to be crucial for the establishment of mature neural circuits. It remains unclear if the peripheral structure of the developing somatosensory system exhibits spontaneous activity, similar to that observed in the retina and cochlea of developing mammals. By establishing an ex vivo calcium imaging system, here we found that neurons in the whisker-innervating region of the trigeminal ganglion (TG) of neonatal mice generate spontaneous activity. A small percentage of neurons showed some obvious correlated activity, and these neurons were mostly located close to one another. TG spontaneous activity was majorly exhibited by medium-to-large diameter neurons, a characteristic of mechanosensory neurons, and was blocked by chelation of extracellular calcium. Moreover, this activity was diminished by the adult stage. Spontaneous activity in the TG during the first postnatal week could be a source of spontaneous activity observed in the neonatal mouse barrel cortex.

BACKGROUND:. Patients with acute bronchospasm can show a distinct slope of the capnogram (“shark fin”) as a result of asynchronous alveolar excretion. Although the slope of the upward alveolar plateau (phase III) in the capnogram waveforms of non-intubated patients is known to help monitor the therapeutic response to acute bronchospasm, little is known about the significance of its slope among intubated patients. Therefore, we quantified the phase III slope of an intubated patient with acute asthma to investigate whether capnogram waveforms could be useful for identifying the response to antibronchospasm treatment in real time. CASE SUMMARY:. The patient was a 53-year-old man who had a history of asthma. He presented to the emergency department with the primary complaint of respiratory distress. He was diagnosed with severe asthma attack and required invasive mechanical ventilation for 10 days, during which we quantified the phase III slope of the capnogram. The phase III slope decreased during treatment, with a significant reduction from the third to the fourth day; however, a significant decrease in end-tidal carbon dioxide (EtCO2) was observed from the fifth to the sixth day. We found that the slope values decreased earlier than EtCO2 reduction, although the absolute EtCO2 values eventually decreased in response to antibronchospasm treatment. CONCLUSION:. There were several reports that evaluated the phase III slope in non-intubated patients with asthma, but this is the first report measuring the phase III slope in an intubated patient over several days. Capnogram waveforms may serve as useful real-time indicators to monitor acute bronchospasm among mechanically ventilated patients.

High temperatures lead to DNA double-strand break accumulation and asynapsis that trigger the meiotic checkpoint and apoptosis of spermatocytes in ex vivo cultures, providing a rationale for the heat sensitivity of sperm production in mammals.

Mouse spermatogenesis, from spermatogonial stem cell proliferation to sperm formation, can be reproduced in vitro by culturing testis tissue masses of neonatal mice. However, it remains to be determined whether this method is also applicable when testis tissues are further divided into tiny fragments, such as segments of the seminiferous tubule (ST), a minimal anatomical unit for spermatogenesis. In this study, we investigated this issue using the testis of an Acrosin-GFP/Histone H3.3-mCherry (Acr/H3) double-transgenic mouse and monitored the expression of GFP and mCherry as indicators of spermatogenic progression. Initially, we noticed that the cut and isolated stretches of ST shrunk rapidly and conglomerated. We therefore maintained the isolation of STs in two ways: segmental isolation without truncation or embedding in soft agarose. In both cases, GFP expression was observed by fluorescence microscopy. By whole-mount immunochemical staining, meiotic spermatocytes and round and elongating spermatids were identified as Sycp3-, crescent-form GFP-, and mCherry-positive cells, respectively. Although the efficiency was significantly lower than that with tissue mass culture, we clearly showed that spermatogenesis can be induced up to the elongating spermatid stage even when the STs were cut into short segments and cultured in isolation. In addition, we demonstrated that lowered oxygen tension was favorable for spermatogenesis both for meiotic progression and for producing elongating spermatids in isolated STs. Culturing isolated STs rather than tissue masses is advantageous for explicitly assessing the various environmental parameters that influence the progression of spermatogenesis.

One of the possible benefits of robot-mediated education is the effect of the robot becoming a catalyst between people and facilitating learning. In this study, the authors focused on an asynchronous active learning method mediated by robots. Active learning is believed to help students continue learning and develop the ability to think independently. Therefore, the authors improved the UGA (User Generated Agent) system that we have created for long-term active learning in COVID-19 to create an environment where children introduce books to each other via robots. The authors installed the robot in an elementary school and conducted an experiment lasting more than a year. As a result, it was confirmed that the robot could continue to be used without getting bored even over a long period of time. They also analyzed how the children created the contents by analyzing the contents that had a particularly high number of views. In particular, the authors observed changes in children’s behavior, such as spontaneous advertising activities, guidance from upperclassmen to lowerclassmen, collaboration with multiple people, and increased interest in technology, even under conditions where the new coronavirus was spreading and children’s social interaction was inhibited.

Human resident memory regulatory T cells (Tregs) exist in the normal, noninflamed skin. Except one, all previous studies analyzed skin Tregs using full-thickness human skin. Considering that thick dermis contains more Tregs than thin epidermis, the current understanding of skin Tregs might be biased toward dermal Tregs. Therefore, we sought to determine the phenotype and function of human epidermal and epithelial Tregs. Human epidermis and epithelium were allowed to float on a medium without adding any exogenous cytokines and stimulations for two days and then emigrants from the explants were analyzed. Foxp3 was selectively expressed in CD4+CD103− T cells in the various human epithelia, as it is highly demethylated. CD4+CD103−Foxp3+ cells suppressed proliferation of other resident memory T cells. The generation and maintenance of epithelial Tregs were independent of hair density and Langerhans cells. Collectively, immune-suppressive CD4+CD103−Foxp3+ Tregs are present in the normal, noninflamed human epidermis and mucosal epithelia.

Tissue-resident memory T cells exist in both the epidermis and the dermis in human skin. To analyze these cells, the skin needs to be incubated with dispase II to separate the two layers, that is, the epidermis and the dermis. The next step varies among researchers; the subsequent enzymatic digestion of the two layers is popular, whereas the spontaneous migration method can also be done. Scraping of these layers to yield skin T cells may reduce antigen modulation. This study aimed to determine each method’s limitations. Dispase II incubation itself cleaves T-cell antigens. Therefore, further enzymatic digestion with collagenases strongly cleaves antigens. The scraping method yields skin T cells that are affected by dispase II as it is. However, skin T-cell yield is low. The spontaneous migration method recovers and/or upregulates antigens with T-cell activation and loses ∼20% of T cells in the floating sheets. However, there was no prominent bias regarding CD103 expression between emigrants and the remaining T cells in the sheets. There were 104 and 105 CD3+ T cells per 1 cm2 of the epidermis and upper dermis, respectively. Collectively, each method has strengths and limitations to analyze both the epidermal and dermal T cells.

Abstract In vitro spermatogenesis (IVS) using air–liquid interphase organ culture method is possible with mouse testis tissues. The same method, however, has been hardly applicable to animals other than mice, only producing no or limited progression of spermatogenesis. In the present study, we challenged IVS of rats with modifications of culture medium, by supplementing chemical substances, including hormones, antioxidants, and lysophospholipids. In addition, reducing oxygen tension by placing tissues in an incubator of lower oxygen concentration and/or applying silicone cover ceiling on top of the tissue were effective for improving the spermatogenic efficiency. Through these modifications of the culture condition, rat spermatogenesis up to round spermatids was maintained over 70 days in the cultured tissue. Present results demonstrated a significant progress in rat IVS, revealing conditions commonly favorable for mice and rats as well as finding rat-specific optimizations. This is an important step towards successful IVS in many animal species, including humans.

Background:. The generation of breast implant–associated anaplastic large-cell lymphoma (BIA-ALCL) is closely associated with textured implants. The phenotype of BIA-ALCL cells is well examined, but its cell of origin remains unknown. Here we investigate what types of T cells are recruited and differentiated in the surrounding capsules and tissues as a consequence of continuous contact with a textured surface. Methods:. Capsule and pericapsule tissues were recovered from patients who had textured or smooth tissue expanders (TEs). These samples were enzymatically digested, and T cells in the samples were analyzed using flow cytometry. Peripheral blood mononuclear cells from the same donors were utilized as a control. Results:. Effector memory CD4+ T cells predominantly infiltrated capsules and tissues without apparent differences between textured and smooth TEs. In these effector memory CD4+ T cells, CD4+ resident memory T cells were generated by smooth TEs but not by textured TEs. However, TNFRSF8/CD30 mRNA expression is higher in the CD69− effector memory CD4+ T cells than in the CD69+ ones. Conclusion:. Textured and smooth TEs differentially recruit and/or differentiate T cells in situ.

Abstract Microbeam radiotherapy (MRT) is based on a spatial fractionation of synchrotron X-ray microbeams at the microscale level. Although the tissue-sparing effect (TSE) in response to non-uniform radiation fields was recognized more than one century ago, the TSE of MRT in the testes and its clinical importance for preventing male fertility remain to be determined. In this study, using the combination of MRT techniques and a unique ex vivo testes organ culture, we show, for the first time, the MRT-mediated TSE for the preservation of spermatogenesis. Furthermore, our high-precision microbeam analysis revealed that the survival and potential migration steps of the non-irradiated germ stem cells in the irradiated testes tissue would be needed for the effective TSE for spermatogenesis. Our findings indicated the distribution of dose irradiated in the testes at the microscale level is of clinical importance for delivering high doses of radiation to the tumor, while still preserving male fertility.

Abstract Purpose Mouse in vitro spermatogenesis is possible with classical organ culture methods, by placing the testis tissue at the interphase between culture medium and air. In this condition, however, a tissue piece tends to round up to be compact, whose central region suffers from shortage of nutrients and oxygen. In this study, the authors improved the culture condition by spreading each tissue thin and flat, by which they were able to get better access to the oxygen and nutrients. Methods Immature mouse testis tissues placed on agarose gel block were forced to spread flat by covering with a polydimethylsiloxane (PDMS) ceiling chip (PC chip). They were then cultured for weeks and evaluated by the transgene expression of Acr‐Gfp, which reflects the progression of spermatogenesis. Results Testis tissues covered with PC chip initiated and maintained spermatogenesis in its wider region than those without PC chip covering. Flow cytometric analysis demonstrated that the PC method yielded more numerous meiotic germ cells than those without PC. Immunohistochemical examination confirmed the authentic histological figure of spermatogenesis from spermatogonia up to round or elongating spermatids. Conclusions The PC chip method is simple and effective to improve the efficiency of in vitro spermatogenesis in the organ culture system.

In vitro spermatogenesis, which produces fertile spermatozoa, has been successfully performed using an organ culture method from murine tissue. Here, we provide a dataset of time-course microarray transcriptome data of in vitro cultured neonate murine testes and age-matched in vivo-derived testes. The dataset presented here is related to the article titled “Transcriptome analysis reveals inadequate spermatogenesis and immediate radical immune reactions during organ culture in vitro spermatogenesis” published in Biochemical and Biophysical Research Communications in 2020 [1]. The raw data and pre-processed data are publicly available on the GEO repository (accession number GSE147982). Furthermore, the dataset provided here includes additional metadata, detailed explanations of the experiment, results of pre-processing, analysis scripts, and lists of differentially expressed genes from in vitro culture testes and in vivo testes at each time point.

Abstract Background Spermatogenesis is one of the most complicated cellular differentiation processes in a body. Researchers struggled to find and develop a micro‐environmental condition that can support the process in vitro. Such endeavors can be traced back to a century ago and are yet continuing. Methods Reports on in vitro spermatogenesis and related works were selected and classified into four categories based on the method used; organ culture, tubule culture, cell culture, and 3‐dimensional cell culture methods. Each report was critically reviewed from the present point of view by authors who have been working on in vitro spermatogenesis with organ culture method over a decade. Results The organ culture method has the longest history and is the most successful method, which produced fertile mouse sperm from spermatogonial stem cells. Formulation of the medium was a key factor, most importantly serum‐derived substances. However, factors in the serum that induce and support spermatogenesis in the cultured tissue remain to be identified. In addition, the success of mouse spermatogenesis is yet to be applied to other animals. On looking into the history of cell culture method, it became clear that Sertoli cells as feeder cells play an important role. Even with Sertoli cells, however, spermatogenic development has been limited to small parts of spermatogenesis, a segmented period of meiotic prophase for instance. Recent developments of organoid or 3‐dimensional culture techniques are promising but they still need further refinements. Conclusion The study of in vitro spermatogenesis progressed significantly over the last century. We need more work, however, to establish a culture system that can induce and maintain complete spermatogenesis of many if not all mammalian species.

Summary: Establishment of precise neuronal connectivity in the neocortex relies on activity-dependent circuit reorganization during postnatal development; however, the nature of cortical activity during this period remains largely unknown. Using two-photon calcium imaging of the barrel cortex in vivo during the first postnatal week, we reveal that layer 4 (L4) neurons within the same barrel fire synchronously in the absence of peripheral stimulation, creating a “patchwork” pattern of spontaneous activity corresponding to the barrel map. By generating transgenic mice expressing GCaMP6s in thalamocortical axons, we show that thalamocortical axons also demonstrate the spontaneous patchwork activity pattern. Patchwork activity is diminished by peripheral anesthesia but is mostly independent of self-generated whisker movements. The patchwork activity pattern largely disappeared during postnatal week 2, as even L4 neurons within the same barrel tended to fire asynchronously. This spontaneous L4 activity pattern has features suitable for thalamocortical (TC) circuit refinement in the neonatal barrel cortex. : By two-photon calcium imaging of layer 4 neurons and thalamocortical axon terminals in neonatal mouse barrel cortex, Mizuno et al. find a patchwork-like spontaneous activity pattern corresponding to the barrel map, which may be important for thalamocortical circuit maturation. Keywords: activity-dependent development, spontaneous activity, synchronized activity, barrel cortex, thalamocortical axons, neonates, in vivo calcium imaging, awake, single-cell labeling, whisker monitoring

As interest continues in sodium reduction technologies, there is a need to understand the changes in physiochemical and sensory characteristics of reduced sodium food products. Previous research has shown that traditionally brewed soy sauce (SS) and fermented flavor enhancer (NFE) offer efficacy as viable sodium reduction ingredients. However, their ability to provide similar results in other meat products with different requirements (e.g., flavor, functionality, etc.) for salt is not well understood. In this study, bacon, beef jerky, summer sausage, and boneless ham treatments were generated with sodium reductions of 30 and 50% by including either SS or NFE alone and in combination with potassium chloride (KCl). Sensory and quality measurements, including salty taste, and product qualities such as color, purge, and texture profiles were evaluated. No differences ( > 0.05) were observed for overall liking of bacon at 30 (SS and NFE) and 50% (SS) reductions containing KCl, and for overall liking of beef jerky (NFE) and boneless ham (SS) at 30% reductions utilizing either SS or NFE as a salt reduction tool. Further, saltiness liking scores showed no change ( > 0.05) at 30% reductions in bacon, beef jerky and boneless ham, while a decreased liking ( < 0.05) were observed in summer sausage products containing SS and NFE. These results suggest the use of SS and NFE can increase perceived salty taste without increasing the sodium content of multiple products investigated. These results indicated that SS and NFE are suitable ingredients to utilize in processed meat products to reduce sodium content, while each product has unique and variable responses in sensory attributes that must be considered.

In the seminiferous tubules of mouse testes, a population of glial cell line-derived neurotrophic factor family receptor alpha 1 (GFRα1)-positive spermatogonia harbors the stem cell functionality and supports continual spermatogenesis, likely independent of asymmetric division or definitive niche control. Here, we show that activation of Wnt/β-catenin signaling promotes spermatogonial differentiation and reduces the GFRα1+ cell pool. We further discovered that SHISA6 is a cell-autonomous Wnt inhibitor that is expressed in a restricted subset of GFRα1+ cells and confers resistance to the Wnt/β-catenin signaling. Shisa6+ cells appear to show stem cell-related characteristics, conjectured from the morphology and long-term fates of T (Brachyury)+ cells that are found largely overlapped with Shisa6+ cells. This study proposes a generic mechanism of stem cell regulation in a facultative (or open) niche environment, with which different levels of a cell-autonomous inhibitor (SHISA6, in this case) generates heterogeneous resistance to widely distributed differentiation-promoting extracellular signaling, such as WNTs.

Battery train is in development as train which can travel in non-electrified section using power supplied from onboard storage system such as lithium-ion battery. However, as characteristics of this type of train, an energy consumption of battery train depends on the state of charge of the storage system. In consideration of these characteristics, we propose a generation method of the timetable which is the most energy-saving when a single battery train travels on a route section containing multiple stations in this research. Although this optimization can be defined as a nonlinear programming problem, we ease to solve this problem using linear approximation to the energy consumption characteristic. In the end, we carried out a simulation in a simple case and achieved 4.7% reduction in energy consumption by the proposed method.

Lipid and fatty acid composition of female Pacific bluefin tuna (PBF, Thunnus orientalis) reproductive and somatic tissues in southwestern North Pacific and Sea of Japan spawning grounds are compared. Total lipid (TL) levels are higher in liver than white muscle tissues. An increased gonadosomatic index (GSI) during the early spawning season coincided with decreased TL. Levels of triacylglycerols (TAG) in PBF liver tissues from the Nansei Islands and Sea of Japan, and white muscle in fishes from the Sea of Japan, decreased during the spawning season, while TAG in ovary tissues did not. Concurrent reductions in TL and increases in GSI early in the spawning season suggest TAG depletion was caused by allocation from liver and white muscle tissues to oocytes, that the liver is one of the important lipid-storage organs in PBF, and this species mostly reliant on capital deposits as a mixed capital-income breeder. Differences of docosahexaenoic acid (DHA) levels between spawning grounds were lower in ovary than in muscle and liver tissues. However, eicosapentaenoic (EPA) and arachidonic acid (ARA) levels that influence egg development and embryo and larval growth are significantly higher in PBF tissues from the Sea of Japan than Nansei Islands, which coincided with larval quality. These suggest a maternal effect exists, with egg quality influencing offspring survival, and that the reproductive strategy of PBF varies according to local variation at each spawning ground.

The magnetic properties of Nd-Fe-B thin films with the three different compositions (#1: Nd12.6Fe81.5B5.9, #2: Nd14.6Fe78.1B7.4 and #3: Nd22.6Fe66.2B11.2) are discussed. With increasing Nd content, the c-axis orientation along the film normal is enhanced. It is found that sample #2 possesses the saturation magnetization Ms very close to that for Nd2Fe14B over a temperature range from 100 to about 300K. The magnetic anisotropy constant Ku2 for sample #2 is the highest among those samples, but smaller by about 20%, as compared to that for Nd2Fe14B. It is of interest to note that the temperature TR at which Ku1 changes its sign is lower by about 30K as compared to that previously reported for Nd2Fe14B. The reason for this discrepancy is not clear, but could be due to the presence of the minority phases of Nd-rich compounds and also a possible contribution of the magneto-elastic effect to the net magnetic anisotropy.

We previously reported the successful induction and completion of mouse spermatogenesis by culturing neonatal testis tissues. The culture medium consisted of α-minimum essential medium (α-MEM), supplemented with Knockout serum replacement (KSR) or AlbuMAX, neither of which were defined chemically. In this study, we formulated a chemically defined medium (CDM) that can induce mouse spermatogenesis under organ culture conditions. It was found that bovine serum albumin (BSA) purified through three different procedures had different effects on spermatogenesis. We also confirmed that retinoic acid (RA) played crucial roles in the onset of spermatogonial differentiation and meiotic initiation. The added lipids exhibited weak promoting effects on spermatogenesis. Lastly, luteinizing hormone (LH), follicle stimulating hormone (FSH), triiodothyronine (T3), and testosterone (T) combined together promoted spermatogenesis until round spermatid production. The CDM, however, was not able to produce elongated spermatids. It was also unable to induce spermatogenesis from the very early neonatal period, before 2 days postpartum, leaving certain factors necessary for spermatogenic induction in mice unidentified. Nonetheless, the present study provided important basic information on testis organ culture and spermatogenesis in vitro.

Mouse spermatogonial stem cells (SSCs) can be cultured for multiplication and maintained for long periods while preserving their spermatogenic ability. Although the cultured SSCs, named germline stem (GS) cells, are targets of genome modification, this process remains technically difficult. In the present study, we tested TALEN and double-nicking CRISPR/Cas9 on GS cells, targeting Rosa26 and Stra8 loci as representative genes dispensable and indispensable in spermatogenesis, respectively. Harvested GS cell colonies showed a high targeting efficiency with both TALEN and CRISPR/Cas9. The Rosa26-targeted GS cells differentiated into fertility-competent sperm following transplantation. On the other hand, Stra8-targeted GS cells showed defective spermatogenesis following transplantation, confirming its prime role in the initiation of meiosis. TALEN and CRISPR/Cas9, when applied in GS cells, will be valuable tools in the study of spermatogenesis and for revealing the genetic mechanism of spermatogenic failure.

A search for early response genes that are activated following germ cell induction from mouse embryonic stem cells in vitro led us to the isolation of a long noncoding RNA that contains a SINE (short interspersed element)-B1F motif that was named R53. In situ hybridization and northern blot analyses revealed that the R53 subfragment RNA bears a B1F motif, is processed from the primary transcript, is expressed in adult testis and is predominantly localized in meiotic metaphase chromatin during spermatogenesis. Recent studies of chromosome-associated RNAs have explored novel functions of noncoding RNAs. Specifically, chromosome-bound noncoding RNAs function not only as structural components of chromosome but also as scaffolds that recruit epigenetic modulators for transcriptional regulation, and they are dynamically rearranged during the cell cycle. However, few studies have explored meiotic chromatin; thus, R53 RNA appears to be the first long noncoding RNA to be tightly associated with the metaphase chromatin during spermatogenesis. Furthermore, R53 knockdown using a lentivirus-mediated RNAi injected into mouse testis and organ culture of the fragments revealed a remarkable reduction in postmeiotic cells and irregular up-regulation of several postmeiotic genes, which suggests the possibility that the SINE-B1-derived noncoding RNA R53 plays an indispensable role in the transcriptional regulation of key spermatogenesis genes.

Background/Aims: Our aim was to identify cognitive factors affecting free recall, cued recall, and recognition tasks in patients with Alzheimer’s disease (AD). Subjects: We recruited 349 consecutive AD patients who attended a memory clinic. Methods: Each patient was assessed using the Alzheimer’s Disease Assessment Scale (ADAS) and the extended 3-word recall test. In this task, each patient was asked to freely recall 3 previously presented words. If patients could not recall 1 or more of the target words, the examiner cued their recall by providing the category of the target word and then provided a forced-choice recognition of the target word with 2 distracters. The patients were divided into groups according to the results of the free recall, cued recall, and recognition tasks. Multivariate logistic regression analysis for repeated measures was carried out to evaluate the net effects of cognitive factors on the free recall, cued recall, and recognition tasks after controlling for the effects of age and recent memory deficit. Results: Performance on the ADAS Orientation task was found to be related to performance on the free and cued recall tasks, performance on the ADAS Following Commands task was found to be related to performance on the cued recall task, and performance on the ADAS Ideational Praxis task was found to be related to performance on the free recall, cued recall, and recognition tasks. Conclusion: The extended 3-word recall test reflects deficits in a wider range of memory and other cognitive processes, including memory retention after interference, divided attention, and executive functions, compared with word-list recall tasks. The characteristics of the extended 3-word recall test may be advantageous for evaluating patients’ memory impairments in daily living.

Research on in vitro spermatogenesis is important for elucidating the spermatogenic mechanism. We previously developed an organ culture method which can support spermatogenesis from spermatogonial stem cells up to sperm formation using immature mouse testis tissues. In this study, we examined whether it is also applicable to mature testis tissues of adult mice. We used two lines of transgenic mice, Acrosin-GFP and Gsg2-GFP, which carry the marker GFP gene specific for meiotic and haploid cells, respectively. Testis tissue fragments of adult GFP mice, aged from 4 to 29 weeks old, which express GFP at full extension, were cultured in medium supplemented with 10% KSR or AlbuMAX. GFP expression decreased rapidly and became the lowest at 7 to 14 days of culture, but then slightly increased during the following culture period. This increase reflected de novo spermatogenesis, confirmed by BrdU labeling in spermatocytes and spermatids. We also used vitamin A-deficient mice, whose testes contain only spermatogonia. The testes of those mice at 13-21 weeks old, showing no GFP expression at explantation, gained GFP expression during culturing, and spermatogenesis was confirmed histologically. In addition, the adult testis tissues of Sl/Sld mutant mice, which lack spermatogenesis due to Kit ligand mutation, were cultured with recombinant Kit ligand to induce spermatogenesis up to haploid formation. Although the efficiency of spermatogenesis was lower than that of pup, present results showed that the organ culture method is effective for the culturing of mature adult mouse testis tissue, demonstrated by the induction of spermatogenesis from spermatogonia to haploid cells.

In general rehabilitation, the walking stick, parallel bars, and walker are used as walking training devices. Users of these devices bear their weight with the hands or legs, and doing so is painful in some cases. In recent years, population aging has progressed rapidly and a labor shortage in the welfare field has become a serious problem. Against this background, it is necessary to develop a walking training device that can motivate patients to train without assistance from a care worker. In this research, we designed and developed a walker equipped with an assist mechanism that translates simulated walking motion into rotational wheel motion. Using the walker, the patient is supported by a saddle, so the hands and legs are relieved of pain due to supporting body weight. The motion transformer is designed using a planar 6-link mechanism, which transforms oscillating motion to rotational motion. Joint angular displacements were measured with a wearable device. Results showed that general walking trajectories can be obtained. This mechanism is designed such that the pedal follows the foot motion in walking by comparing the coupler curve of the mechanism with the trajectory of an ankle joint in a walking motion. We manufactured four types of walkers for testing. Type I uses a motion transformer consisting of only revolute pairs. Type II uses a motion transformer consisting of revolute pairs and a prismatic pair (sliding joint). Type II-ankle free uses a Type II motion transformer from which two links were removed in order to allow free rotational motion of the ankle joint. Lastly, the Type III walker has an axle in the front in order to facilitate riding from the rear and to allow free rotational motion of the ankle joint. Pedaling force was measured and compared among the four types. It was found that free ankle joint movement was effective in decreasing the pedaling force. The results indicate that the motion transformer could be driven with a small force and with a simulated walking motion as the input. However, the angle between the direction of foot velocity and the direction of pedaling force was approximately π/2, and so driving the device using a pedaling motion could produce a sensation different from that of a normal walking motion. Eliminating discomfort during operation is left as a future task.

A plug-based microfluidic system based on the principle of the light-addressable potentiometric sensor (LAPS) is proposed. The LAPS is a semiconductor-based chemical sensor, which has a free addressability of the measurement point on the sensing surface. By combining a microfluidic device and LAPS, ion sensing can be performed anywhere inside the microfluidic channel. In this study, the sample solution to be measured was introduced into the channel in a form of a plug with a volume in the range of microliters. Taking advantage of the light-addressability, the position of the plug could be monitored and pneumatically controlled. With the developed system, the pH value of a plug with a volume down to 400 nL could be measured. As an example of plug-based operation, two plugs were merged in the channel, and the pH change was detected by differential measurement.

Incorporation of bovine serum-derived albumin formulation (AlbuMAX) into a basic culture medium, MEMa, enables the completion of in vitro spermatogenesis through testicular tissue culture in mice. However, this medium was not effective in other animals. Therefore, we sought an alternative approach for in vitro spermatogenesis using a synthetic medium without AlbuMAX and aimed to identify its essential components. In addition to factors known to be important for spermatogenesis, such as retinoic acid and reproductive hormones, we found that antioxidants (vitamin E, vitamin C, and glutathione) and lysophospholipids are vital for in vitro spermatogenesis. Moreover, based on our experience with microfluidic devices (MFD), we developed an alternative approach, the PDMS-ceiling method (PC method), which involves simply covering the tissue with a flat chip made of PDMS, a silicone resin material used in MFD. The PC method, while straightforward, integrates the advantages of MFD, enabling improved and uniform oxygen and nutrient supply via tissue flattening. Furthermore, our studies underscored the significance of lowering the oxygen concentration to 10-15%. Using an integrated cultivation method based on these findings, we successfully achieved in vitro spermatogenesis in rats, which has been a long-standing challenge. Further improvements in culture conditions would pave the way for spermatogenesis completion in diverse animal species. [ABSTRACT FROM AUTHOR]