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The adaptor protein GAREM has two subtypes. Each is involved in Erk activation signaling downstream of the cell growth factor receptor in cultured cells. Regarding their role in individual animals, we have previously reported that mice deficient in GAREM2, which is highly expressed in the brain, exhibit emotional changes. In this paper, we report an amino acid substitution mutation (K291R) in GAREM1, in a patient with idiopathic short stature, which indicates that the mutant exhibits dominant-negative properties. The GAREM K291R mutant did not promote Erk activation in EGF-stimulated cultured cells. Similar features were also observed in cells in which GAREM1 expression was suppressed by genome editing; along with Erk, phosphorylation of S6 kinase and 4EBP1, whose activation is necessary for cell proliferation and biological growth, were inhibited Furthermore, we generated mice deficient in GAREM1 and showed that the mutant mice are lighter in weight. Overall, the results of this paper suggest that GAREM1 is required for normal growth and for maintaing average body size in humans and mice.

The homeostatic regulation of sleep is characterized by rebound sleep after prolonged wakefulness, but the molecular and cellular mechanisms underlying this regulation are still unknown. We show here that CaMKII-dependent activity control of parvalbumin (PV)-expressing cortical neurons is involved in sleep homeostasis regulation. Prolonged wakefulness enhances cortical PV-neuron activity. Chemogenetic suppression or activation of cortical PV neurons inhibits or induces rebound sleep, implying that rebound sleep is dependent on increased activity of cortical PV neurons. Furthermore, we discovered that CaMKII kinase activity boosts the activity of cortical PV neurons, and that kinase activity is important for homeostatic sleep rebound. We propose that CaMKII-dependent PV-neuron activity represents negative feedback inhibition of cortical neural excitability, which serves as the distributive cortical circuits for sleep homeostatic regulation.

Genomes of higher eukaryotes encode many uncharacterized proteins, and the functions of these proteins cannot be predicted from the primary sequences due to a lack of conserved functional domains. In this study, we focused on a poorly characterized protein UGS148 that is highly expressed in a specialized cell type called tanycytes that line the ventral wall of the third ventricle in the hypothalamus. Immunostaining of UGS148 revealed the fine morphology of tanycytes with highly branched apical ER membranes. Immunoprecipitation revealed that UGS148 associated with mitochondrial ATPase at least in vitro, and ER and mitochondrial signals occasionally overlapped in tanycytes. Mutant mice lacking UGS148 did not exhibit overt phenotypes, suggesting that UGS148 was not essential in mice reared under normal laboratory conditions. We also found that RNA probes that were predicted to uniquely detect UGS148 mRNA cross-reacted with uncharacterized RNAs, highlighting the importance of experimental validation of the specificity of probes during the hybridization-based study of RNA localization.

Background: Early neonates of both large and small mammals are able to regenerate the myocardium through cardiomyocyte proliferation for only a short period after birth. This myocardial regenerative capacity declines in parallel with withdrawal of cardiomyocytes from the cell cycle in the first few postnatal days. No mammalian species examined to date has been found capable of a meaningful regenerative response to myocardial injury later than 1 week after birth. Methods: We examined cardiomyocyte proliferation in neonates of the marsupial opossum ( Monodelphis domestica ) by immunostaining at various times after birth. The regenerative capacity of the postnatal opossum myocardium was assessed after either apex resection or induction of myocardial infarction at postnatal day 14 or 29, whereas that of the postnatal mouse myocardium was assessed after myocardial infarction at postnatal day 7. Bioinformatics data analysis, immunofluorescence staining, and pharmacological and genetic intervention were applied to determine the role of AMPK (5′-AMP–activated protein kinase) signaling in regulation of the mammalian cardiomyocyte cell cycle. Results: Opossum neonates were found to manifest cardiomyocyte proliferation for at least 2 weeks after birth at a frequency similar to that apparent in early neonatal mice. Moreover, the opossum heart at postnatal day 14 showed substantial regenerative capacity both after apex resection and after myocardial infarction injury, whereas this capacity had diminished by postnatal day 29. Transcriptomic and immunofluorescence analyses indicated that AMPK signaling is activated in postnatal cardiomyocytes of both opossum and mouse. Pharmacological or genetic inhibition of AMPK signaling was sufficient to extend the postnatal window of cardiomyocyte proliferation in both mouse and opossum neonates as well as of cardiac regeneration in neonatal mice. Conclusions: The marsupial opossum maintains cardiomyocyte proliferation and a capacity for myocardial regeneration for at least 2 weeks after birth. As far as we are aware, this is the longest postnatal duration of such a capacity among mammals examined to date. AMPK signaling was implicated as an evolutionarily conserved regulator of mammalian postnatal cardiomyocyte proliferation.

Contemplando a realidade contemporânea, a Revista EntreLínguas traz artigos com reflexões e contribuições relevantes da grande área de Letras e Linguística em um cenário de transição com inúmeros desafios. A importância de uma formação compreensiva e atualizada, tanto para professores em exercício como para novos professores, torna-se primordial para compreender, respeitar e lidar com diversidades, linguagens em novos espaços educacionais. Assim, esta edição da Revista EntreLínguas apresenta diversos artigos que abordam o tema do ensino, das letras e da linguística sob diversos olhares e análises, estando presentes nesse compêndio trabalhos das universidades: N.A. Dobrolyubov Nizhny Novgorod State Linguistic University, Kazan Federal University, Xi’na Internacional Studies University, Firat University, Ferdowsi University of Mashhad, American University of Central Asia, Azerbaijan State Pedagogical University, Korkyt ATA Kyzylorda State University, Mahambet Utemisov West Kazakhstan State University, Agrarian University of Havana, Kyrgyz-Turkish University, Abdu Vali Khan University, Sichuan International Studies University, Saint Petersburg State University, L.N. Gumilov Eurasian National University, North-Eastern Federal University, University of Presov e RUDN University.

O objetivo deste artigo é tecer algumas considerações e reflexões a respeito do senso de plausibilidade do professor, especificamente para fins acadêmicos, num contexto de aulas em ambiente online síncrono. Para tanto, primeiramente discorreremos acerca do conceito do senso de plausibilidade. Em segundo lugar, abordaremos a temática do ensino e aprendizagem de línguas que utilizam ferramentas e/ou plataformas online, bem como a definição do conceito de EAD (ensino a distância), além de discutir a respeito da definição da noção de IFA (Inglês para Fins Acadêmicos). Posteriormente, focalizaremos questões envolvendo relatos de experiências de cursos de IFA em ambiente EAD. E por fim, faremos considerações com vistas a apontar possíveis problemáticas e encaminhamentos no que diz respeito à abordagem do ensino de inglês para fins acadêmicos em contextos EAD

Based on the concepts of internationalization and foreign language proficiency, in this article, we aim to map the data on proficiency levels of learners who took part in English teaching projects supported by diverse funding sources that have been developed in a public university in Sao Paulo state. Within these projects, courses and online placement tests are offered to undergraduate and graduate students, as well as for members of the administrative staff. The courses are online (synchronous) in the web conferencing tool Zoom. The teachers in these courses are undergraduate and graduate students in the area of Language and Linguistics and they are supervised by professors from different areas of expertise, such as teachers’ education, technology in language teaching and language assessment.

Background: The maternal circulatory system and hormone balance both change dynamically during pregnancy, delivery, and the postpartum period. Although atrial natriuretic peptides and brain natriuretic peptides produced in the heart control circulatory homeostasis through their common receptor, NPR1, the physiologic and pathophysiologic roles of endogenous atrial natriuretic peptide/brain natriuretic peptide in the perinatal period are not fully understood. Methods: To clarify the physiologic and pathophysiologic roles of the endogenous atrial natriuretic peptide/brain natriuretic peptide–NPR1 system during the perinatal period, the phenotype of female wild-type and conventional or tissue-specific Npr1-knockout mice during the perinatal period was examined, especially focusing on maternal heart weight, blood pressure, and cardiac function. Results: In wild-type mice, lactation but not pregnancy induced reversible cardiac hypertrophy accompanied by increases in fetal cardiac gene mRNAs and ERK1/2 (extracellular signaling-regulated kinase) phosphorylation. Npr1-knockout mice exhibited significantly higher plasma aldosterone level than did wild-type mice, severe cardiac hypertrophy accompanied by fibrosis, and left ventricular dysfunction in the lactation period. Npr1-knockout mice showed a high mortality rate over consecutive pregnancy–lactation cycles. In the hearts of Npr1-knockout mice during or after the lactation period, an increase in interleukin-6 mRNA expression, phosphorylation of signal transducer and activator of transcription 3, and activation of the calcineurin–nuclear factor of the activated T cells pathway were observed. Pharmacologic inhibition of the mineralocorticoid receptor or neuron-specific deletion of the mineralocorticoid receptor gene significantly ameliorated cardiac hypertrophy in lactating Npr1-knockout mice. Anti–interleukin-6 receptor antibody administration tended to reduce cardiac hypertrophy in lactating Npr1-knockout mice. Conclusions: These results suggest that the characteristics of lactation-induced cardiac hypertrophy in wild-type mice are different from exercise-induced cardiac hypertrophy, and that the endogenous atrial natriuretic peptide/brain natriuretic peptide–NPR1 system plays an important role in protecting the maternal heart from interleukin-6–induced inflammation and remodeling in the lactation period, a condition mimicking peripartum cardiomyopathy.

Hiroyuki Kondo, Takafumi Matsumura, Mari Kaneko, Kenichi Inoue, Hidetaka Kosako, Masahito Ikawa, Yousuke Takahama, Izumi Ohigashi, PITHD1 is a proteasome-interacting protein essential for male fertilization, Journal of Biological Chemistry, Volume 295, Issue 6, 2020, Pages 1658-1672, ISSN 0021-9258, https://doi.org/10.1074/jbc.RA119.011144., The proteasome is a protein-degrading molecular complex that is necessary for protein homeostasis and various biological functions, including cell cycle regulation, signal transduction, and immune response. Proteasome activity is finely regulated by a variety of proteasome-interacting molecules. PITHD1 is a recently described molecule that has a domain putatively capable of interacting with the proteasome. However, it is unknown whether PITHD1 can actually bind to proteasomes and what it does in vivo. Here we report that PITHD1 is detected specifically in the spermatids in the testis and the cortical thymic epithelium in the thymus. Interestingly, PITHD1 associates with immunoproteasomes in the testis, but not with thymoproteasomes in the thymus. Mice deficient in PITHD1 exhibit severe male infertility accompanied with morphological abnormalities and impaired motility of spermatozoa. Furthermore, PITHD1 deficiency reduces proteasome activity in the testis and alters the amount of proteins that are important for fertilization capability by the sperm. However, the PITHD1-deficient mice demonstrate no detectable defects in the thymus, including T cell development. Collectively, our results identifyPITHD1as a proteasome- interacting protein that plays a nonredundant role in the male reproductive system.

Skeletal muscle is known to regulate bone homeostasis through muscle-bone interaction, although factors that control this activity remain unclear. Here, we newly established Smad3-flox mice, and then generated skeletal muscle-specific Smad2/Smad3 double conditional knockout mice (DcKO) by crossing Smad3-flox with skeletal muscle-specific Ckmm Cre and Smad2-flox mice. We show that immobilization-induced gastrocnemius muscle atrophy occurring due to sciatic nerve denervation was partially but significantly inhibited in DcKO mice, suggesting that skeletal muscle cell-intrinsic Smad2/3 is required for immobilization-induced muscle atrophy. Also, tibial bone atrophy seen after sciatic nerve denervation was partially but significantly inhibited in DcKO mice. Bone formation rate in wild-type mouse tibia was significantly inhibited by immobilization, but inhibition was abrogated in DcKO mice. We propose that skeletal muscle regulates immobilization-induced bone atrophy via Smad2/3, and Smad2/3 represent potential therapeutic targets to prevent both immobilization-induced bone and muscle atrophy.

The reduced sleep duration observed in Camk2a and Camk2b knockout mice revealed the role of Ca2+/calmodulin-dependent protein kinase II (CaMKII)α/CAMKIIβ as sleep-promoting kinases and lead to the phosphorylation hypothesis of sleep. However, the underlying mechanism of sleep regulation by kinases and protein phosphorylation is largely unknown. Here, we demonstrate that the phosphorylation states of CaMKIIβ regulates sleep duration and sleep needs. Importantly, the activation or inhibition of CaMKIIβ can increase or decrease sleep duration by almost two-fold, supporting the role of CaMKIIβ as a core sleep regulator in mammals. This sleep regulation depends on the kinase activity of CaMKIIβ in excitatory neurons. Furthermore, CaMKIIβ mutants mimicking different phosphorylation states can regulate various sleep steps including sleep induction, sleep maintenance, and sleep cancelation. Key CaMKIIβ residues responsible for the mode switch undergo ordered (auto-)phosphorylation. We thus propose that ordered multi-site phosphorylation of CaMKIIβ underlies multi-step sleep regulation in mammals.

Genomes of higher eukaryotes encode many uncharacterized proteins, and the functions of these proteins cannot be predicted from the primary sequences due to a lack of conserved functional domains. During a screening of novel noncoding RNAs abundantly expressed in mouse brains, we incidentally identified a gene termed Tanmp, which encoded an endoplasmic reticulum (ER) protein without known functional domains. Tanmp is specifically expressed in the nervous system, and the highest expression was observed in a specialized cell type called tanycyte that aligns the ventral wall of the third ventricle in the hypothalamus. Immunostaining of Tanmp revealed the fine morphology of tanycytes with highly branched apical ER membranes. Immunoprecipitation revealed that Tanmp associates with mitochondrial ATPase at least in vitro, and ER and mitochondrial signals occasionally overlapped in tanycytes. Mutant mice lacking Tanmp did not exhibit overt phenotypes, suggesting that Tanmp is not essential in mice reared under normal laboratory conditions. We also found that RNA probes that are predicted to uniquely detect Tanmp mRNA cross-reacted with uncharacterized RNAs, highlighting the importance of experimental validation of the specificity of probes during the hybridization-based study of RNA localization.

The pathophysiological role of mammalian target of rapamycin complex 1 (mTORC1) in neurodegenerative diseases is established, but possible therapeutic targets responsible for its activation in neurons must be explored. Here we identified solute carrier family 38a member 1 (SNAT1, Slc38a1) as a positive regulator of mTORC1 in neurons. Slc38a1flox/flox and Synapsin I-Cre mice were crossed to generate mutant mice in which Slc38a1 was selectively deleted in neurons. Measurement of 2,3,5-triphenyltetrazolium chloride (TTC) or the MAP2-negative area in a mouse model of middle cerebral artery occlusion (MCAO) revealed that Slc38a1 deficiency decreased infarct size. We found a transient increase in the phosphorylation of p70S6k1 (pp70S6k1) and a suppressive effect of rapamycin on infarct size in MCAO mice. Autophagy inhibitors completely mitigated the suppressive effect of SNAT1 deficiency on neuronal cell death under in vitro stroke culture conditions. These results demonstrate that SNAT1 promoted ischemic brain damage via mTOR-autophagy system., Yamada et al. demonstrate that solute carrier family 38a member 1 (SNAT1, Slc38a1) is as a positive regulator of mTORC1 that promotes neuronal cell death, using neuron-specific Slc38a1 knockout mice. This study shows that SNAT1 exacerbates ischemic brain damage through mTOR-mediated suppression of autophagy.

O cenário educacional brasileiro tem vivenciado políticas linguísticas que primam pela melhoria da proficiência em língua estrangeira no Ensino Superior. Com a globalização e internacionalização, a universidade deve reconhecer a importância do desenvolvimento da proficiência linguística, tendo em vista o acesso ao conhecimento produzido internacionalmente e sua inserção internacional (ABREU-E-LIMA et al., 2016). A universidade desempenha um papel primordial na implementação de ações pedagógicas para oferecer suporte e articulação para tais ações. O presente estudo contextualiza o Idiomas sem Fronteiras, um programa nacional do Ministério da Educação voltado para o ensino/aprendizagem de línguas estrangeiras para alunos, pesquisadores e servidores de instituições públicas. Serão abordadas as especificidades da UNESP, detalhando sua implementação.

AimsSecreted factors produced by adipose tissue are involved in the pathogenesis of cardiovascular disease. We previously identified adipolin, also known as C1q/TNF-related protein 12, as an insulin-sensitizing adipokine. However, the role of adipolin in vascular disease remains unknown. Here, we investigated whether adipolin modulates pathological vascular remodelling.Methods and resultsAdipolin-knockout (APL-KO) and wild-type (WT) mice were subjected to wire-induced injury of the femoral artery. APL-KO mice showed increased neointimal thickening after vascular injury compared with WT mice, which was accompanied by an enhanced inflammatory response and vascular cell proliferation in injured arteries. Adipolin deficiency also led to a reduction in transforming growth factor-β (TGF-β) 1 protein levels in injured arteries. Treatment of cultured macrophages with adipolin protein led to a reduction in lipopolysaccharide-stimulated expression of inflammatory mediators, including tumour necrosis factor (TNF)-α, interleukin (IL) 6, and monocyte chemotactic protein (MCP)-1. These effects were reversed by inhibition of TGF-β receptor II (TGF-βRII)/Smad2 signalling. Adipolin also reduced platelet-derived growth factor (PDGF)-BB-stimulated proliferation of vascular smooth muscle cells (VSMCs) through a TGF-βRII/Smad2-dependent pathway. Furthermore, adipolin treatment significantly increased TGF-β1 concentration in media from cultured VSMCs and macrophages.ConclusionThese data indicate that adipolin protects against the development of pathological vascular remodelling by attenuating macrophage inflammatory responses and VSMC proliferation.

Osteoblasts arise from bone-surrounding connective tissue containing tenocytes and fibroblasts. Lineages of these cell populations and mechanisms of their differentiation are not well understood. Screening enhancer-trap lines of zebrafish allowed us to identify Ebf3 as a transcription factor marking tenocytes and connective tissue cells in skeletal muscle of embryos. Knockout of Ebf3 in mice had no effect on chondrogenesis but led to sternum ossification defects as a result of defective generation of Runx2+ pre-osteoblasts. Conditional and temporal Ebf3 knockout mice revealed requirements of Ebf3 in the lateral plate mesenchyme cells (LPMs), especially in tendon/muscle connective tissue cells, and a stage-specific Ebf3 requirement at embryonic day 9.5-10.5. Upregulated expression of connective tissue markers, such as Egr1/2 and Osr1, increased number of Islet1+ mesenchyme cells, and downregulation of gene expression of the Runx2 regulator Shox2 in Ebf3-deleted thoracic LPMs suggest crucial roles of Ebf3 in the onset of lateral plate mesoderm differentiation towards osteoblasts forming sternum tissues., Highlighted Article: Analysis of conditional and temporal knockout mice reveals roles of Ebf3 in the differentiation of lateral plate mesenchymal cells towards pre-osteoblasts during sternum ossification at the boundary of the lateral and somitic mesoderm.

The multi-subunit eukaryotic CCR4-NOT complex imparts gene expression control primarily via messenger RNA (mRNA) decay. Here, we present the role of subunit CNOT9 in target mRNA decay during embryonic development. CNOT9 null mice appear normal by the onset of gastrulation (E7.0), however, exhibit growth and differentiation defects accompanied by extensive cell death by embryonic day 9.5 (E9.5). Sox-2 Cre conditional CNOT9 knockout mice show almost identical phenotype with brief delay in onset and progression, suggesting defects to be epiblast-dominant. Among various identified targets, we show thatLefty2mRNA expression is post-transcriptionally regulated by CNOT9.Lefty2 3’-UTRcontaining mRNA has significantly higher stability in cells expressing mutant form of CNOT9, relative to cells expressing wild-type CNOT9. In addition, CNOT9 primarily localizes within the cytoplasm and bridges interactions between the CCR4-NOT complex and miRNA-RISC complex in gastrulating embryos.

Summary Marsupials represent one of three extant mammalian subclasses with very unique characteristics not shared by other mammals. Most notably, much of the development of neonates immaturely born after a relatively short gestation takes place in the external environment. Among marsupials, the gray short-tailed opossum (Monodelphis domestica; hereafter "the opossum") is one of very few established laboratory models. Due to many biologically unique characteristics and experimentally advantageous features, the opossum is used as a prototype species for basic research on marsupial biology.1,2 However, in vivo studies of gene function in the opossum, and thus marsupials in general, lag far behind those of eutherian mammals due to the lack of reliable means to manipulate their genomes. In this study, we describe the successful generation of genome edited opossums by a combination of refined methodologies in reproductive biology and embryo manipulation. We took advantage of the opossum's resemblance to popular rodent models, such as the mouse and rat, in body size and breeding characteristics. First, we established a tractable pipeline of reproductive technologies, from induction of ovulation, timed copulation, and zygote collection to embryo transfer to pseudopregnant females, that warrant an essential platform to manipulate opossum zygotes. Further, we successfully demonstrated the generation of gene knockout opossums at the Tyr locus by microinjection of pronuclear stage zygotes using CRISPR/Cas9 genome editing, along with germline transmission of the edited alleles to the F1 generation. This study provides a critical foundation for venues to expand mammalian reverse genetics into the metatherian subclass.

Mutations in the X-linked gene Protocadherin-19 (Pcdh19) cause female-limited epilepsy and mental retardation in humans. Although Pcdh19 is known to be a homophilic cell-cell adhesion molecule, how its mutations bring about female-specific disorders remains elusive. Here, we report the effects of Pcdh19 knockout in mice on their development and behavior. Pcdh19 was expressed in various brain regions including the cerebral cortex and hippocampus. Although Pcdh19-positive cells were evenly distributed in layer V of wild-type cortices, their distribution became a mosaic in Pcdh19 heterozygous female cortices. In cortical and hippocampal neurons, Pcdh19 was localized along their dendrites, showing occasional accumulation on synapses. Pcdh19 mutants, however, displayed no detectable abnormalities in dendrite and spine morphology of layer V neurons. Nevertheless, Pcdh19 hemizygous males and heterozygous females showed impaired behaviors including activity defects under stress conditions. Notably, only heterozygous females exhibited decreased fear responses. In addition, Pcdh19 overexpression in wild-type cortices led to ectopic clustering of Pcdh19-positive neurons. These results suggest that Pcdh19 is required for behavioral control in mice, but its genetic loss differentially affects the male and female behavior, as seen in human, and they also support the hypothesis that the mosaic expression of Pcdh19 in brains perturbs neuronal interactions.

TRIOBP remodels the cytoskeleton by forming unusually dense F-actin bundles and is implicated in human cancer, schizophrenia, and deafness. Mutations ablating human and mouse TRIOBP-4 and TRIOBP-5 isoforms are associated with profound deafness, as inner ear mechanosensory hair cells degenerate after stereocilia rootlets fail to develop. However, the mechanisms regulating formation of stereocilia rootlets by each TRIOBP isoform remain unknown. Using 3 new Triobp mouse models, we report that TRIOBP-5 is essential for thickening bundles of F-actin in rootlets, establishing their mature dimensions and for stiffening supporting cells of the auditory sensory epithelium. The coiled-coil domains of this isoform are required for reinforcement and maintenance of stereocilia rootlets. A loss of TRIOBP-5 in mouse results in dysmorphic rootlets that are abnormally thin in the cuticular plate but have increased widths and lengths within stereocilia cores, and causes progressive deafness recapitulating the human phenotype. Our study extends the current understanding of TRIOBP isoform-specific functions necessary for life-long hearing, with implications for insight into other TRIOBPopathies.

Background: This study evaluated the characteristics of subependymal giant cell astrocytoma (SEGA) in patients with tuberous sclerosis complex (TSC) entered into the TuberOus SClerosis registry to increase disease Awareness (TOSCA). Methods: The study was conducted at 170 sites across 31 countries. Data from patients of any age with a documented clinical visit for TSC in the 12 months preceding enrollment or those newly diagnosed with TSC were entered. Results: SEGA were reported in 554 of 2,216 patients (25%). Median age at diagnosis of SEGA was 8 years (range, 18 years. SEGA were symptomatic in 42.1% of patients. Symptoms included increased seizure frequency (15.8%), behavioural disturbance (11.9%), and regression/loss of cognitive skills (9.9%), in addition to those typically associated with increased intracranial pressure. SEGA were significantly more frequent in patients with TSC2 compared to TSC1 variants (33.7 vs. 13.2 %, p < 0.0001). Main treatment modalities included surgery (59.6%) and mammalian target of rapamycin (mTOR) inhibitors (49%). Conclusions: Although SEGA diagnosis and growth typically occurs during childhood, SEGA can occur and grow in both infants and adults.

Summary Live imaging is one of the most powerful technologies for studying the behaviors of cells and molecules in living embryos. Previously, we established a series of reporter mouse lines in which specific organelles are labeled with various fluorescent proteins. In this study, we examined the localizations of fluorescent signals during preimplantation development of these mouse lines, as well as a newly established one, by time‐lapse imaging. Each organelle was specifically marked with fluorescent fusion proteins; fluorescent signals were clearly visible during the whole period of time‐lapse observation, and the expression of the reporters did not affect embryonic development. We found that some organelles dramatically change their sub‐cellular distributions during preimplantation stages. In addition, by crossing mouse lines carrying reporters of two distinct colors, we could simultaneously visualize two types of organelles. These results confirm that our reporter mouse lines can be valuable genetic tools for live imaging of embryonic development.

High-fat diet (HFD) induces low-grade chronic inflammation and insulin resistance. However, little is known about the mechanism underlying HFD-induced chronic inflammation in peripheral insulin-responsive tissues. Here, we show that colonic pro-inflammatory macrophages regulate insulin sensitivity under HFD conditions. To investigate the pathophysiological role of colonic macrophages, we generated macrophage-specific chemokine (C-C Motif) receptor 2 (Ccr2) knockout (M-Ccr2KO) and intestinal epithelial cell-specific tamoxifen-inducible Ccl2 knockout (Vil-Ccl2KO) mice. Both strains exhibited similar body weight to control under HFD. However, they exhibited decreased infiltration of colonic pro-inflammatory macrophages, decreased intestinal permeability, and inactivation of the colonic inflammasome. Interestingly, they showed significantly improved glucose tolerance and insulin sensitivity with decreased chronic inflammation of adipose tissue. Therefore, inhibition of pro-inflammatory macrophage infiltration prevents HFD-induced insulin resistance and could be a novel therapeutic approach for type 2 diabetes.

Polarized epithelial cells exhibit a characteristic array of microtubules that are oriented along the apicobasal axis of the cells. The minus-ends of these microtubules face apically, and the plus-ends face toward the basal side. The mechanisms underlying this epithelial-specific microtubule assembly remain unresolved, however. Here, using mouse intestinal cells and human Caco-2 cells, we show that the microtubule minus-end binding protein CAMSAP3 (calmodulin-regulated-spectrin-associated protein 3) plays a pivotal role in orienting the apical-to-basal polarity of microtubules in epithelial cells. In these cells, CAMSAP3 accumulated at the apical cortices, and tethered the longitudinal microtubules to these sites. Camsap3 mutation or depletion resulted in a random orientation of these microtubules; concomitantly, the stereotypic positioning of the nucleus and Golgi apparatus was perturbed. In contrast, the integrity of the plasma membrane was hardly affected, although its structural stability was decreased. Further analysis revealed that the CC1 domain of CAMSAP3 is crucial for its apical localization, and that forced mislocalization of CAMSAP3 disturbs the epithelial architecture. These findings demonstrate that apically localized CAMSAP3 determines the proper orientation of microtubules, and in turn that of organelles, in mature mammalian epithelial cells.

Holocrine secretion is a specific mode of secretion involving secretion of entire cytoplasmic materials with remnants of dead cells, as observed in multicellular exocrine glands of reptiles, birds, and mammals. Here, we found that sebaceous glands in mice, representative of multicellular exocrine glands of mammals, exhibit a form of polarized stratified epithelium equipped with tight junctions (TJs), and found that holocrine secretion occurred outside the TJ barriers. Sebaceous glands share characteristics of stratified epithelia with interfollicular epidermis, including basal-layer-restricted cell proliferation, TJ barrier formation at a specific single layer of cells with apico-basolateral plasma membrane polarity, and cell death outside the TJ barrier. Knockout of claudin-1, a transmembrane adhesive protein in TJs, in mice caused leakage of the TJ barrier in sebaceous glands and incomplete degradation of the plasma membrane and nuclei during holocrine secretion. Claudin-1 knockout resulted in the accumulation of incompletely degenerated sebocytes in sebaceous ducts, suggesting that the TJ barrier was necessary for differentiation of holocrine secretion. The redefinition of sebaceous glands as TJ-forming stratified epithelia provides an important framework to understand the molecular mechanism of holocrine secretion.

During orthodontic treatment, binding teeth, may change the topographically organized representation of teeth in the cerebral cortex. To test the hypothesis that experimental tooth movement (ETM) changes the somatotopy of an individual tooth arrangement in the somatosensory cortex, we examined the spatiotemporal features of cortical excitatory propagation in response to mechanical stimulation of the maxillary incisor or molar using optical imaging in late adolescent rats without or with ETM. The ETM models consisted of 1d, 3d, and 7d ETM in which a closed-coil spring was ligated between the maxillary first molar and incisors. In controls, incisor and molar mechanical stimulation evoked excitation in the rostral and dorsocaudal regions of the primary somatosensory cortex (S1), respectively. In addition, the secondary somatosensory cortex and insular oral region (S2/IOR) were also activated. Incisor stimulation-induced excitatory regions in S1 of 3d and 7d ETM shifted without changing the maximum excitatory area or peak amplitude; the incisor stimulation-responding region moved toward the dorsocaudal region, which responded to molar stimulation in the control. This shift in excitatory region was not observed in 1d ETM. One day after removal of the coil spring that was attached for 6 days, the excitatory region shift in S1 was recovered to the control region. On the other hand, 1d ETM exhibited facilitation of the excitatory area and peak amplitude upon molar stimulation, and the facilitation of excitatory propagation disappeared in 3d and 7d ETM. These results may explain the clinical finding that abnormal sensation temporally occurs during orthodontic treatment.

The primary sensory cortex exhibits neuroplastic changes responding to sensory disturbances, and GABAergic synaptic transmission plays a critical role in the regulation of plasticity. The insular cortex (IC) integrates orofacial nociceptive signals conveyed via myelinated Aδ- and unmyelinated C-fibers. However, it has been unknown whether a disturbance of nociceptive inputs, such as a deletion of the peripheral nerves, alters GABAergic local circuit in IC. The present study elucidated GABAergic synaptic transmission in the model rat whose C-fibers were ablated by capsaicin injection 1–2 days after birth. In vivo optical imaging revealed that capsaicin-treated rats showed a facilitative excitatory propagation in IC responding to dental pulp stimulation. Whole-cell patch-clamp recording from pyramidal neurons (Pyr) demonstrated that capsaicin-treated rats showed the smaller amplitude of miniature inhibitory postsynaptic currents (IPSCs) than sham-treated rats without changing the frequency. Furthermore, replacement of extracellular Ca2+ to Sr2+, which induces an asynchronous release of neurotransmitters in the quantal size, induced a smaller amplitude of asynchronous unitary IPSCs recorded from fast-spiking GABAergic interneuron to Pyr connections in capsaicin-treated rats than sham-treated rats. These results suggest that capsaicin treatment depresses IPSCs via a postsynaptic mechanism. To confirm this possibility, the variance-mean analysis of unitary IPSCs was employed and we found that quantal size of GABAergic synaptic transmission was smaller in capsaicin-treated rats than in sham-treated rats. These results suggest that ablation of C-fibers induces plastic changes in GABAergic synaptic transmission by decreasing postsynaptic GABAA receptor-mediated conductance, which is a possible mechanism of the facilitative excitation in IC of capsaicin-treated rats.

In addition to thymic stromal lymphopoietin and IL-33, IL-25 is known to induce TWe investigated the contribution of IL-25 to CHS using Il25CHS was evaluated by means of measurement of ear skin thickness in mice after fluorescein isothiocyanate painting. Skin dendritic cell (DC) migration, hapten-specific TIn contrast to thymic stromal lymphopoietin, we found that IL-25 was not essential for skin DC migration or hapten-specific TOur results identify a novel IL-25 inflammatory pathway involved in induction of T

Muscle spindles in the jaw-closing muscles, which are innervated by trigeminal mesencephalic neurons (MesV neurons), control the strength of occlusion and the position of the mandible. The mechanisms underlying cortical processing of proprioceptive information are critical to understanding how sensory information from the masticatory muscles regulates orofacial motor function. However, these mechanisms are mostly unknown. The present study aimed to identify the regions that process proprioception of the jaw-closing muscles using in vivo optical imaging with a voltage-sensitive dye in rats under urethane anesthesia. First, jaw opening that was produced by mechanically pulling down the mandible evoked an optical response, which reflects neural excitation, in two cortical regions: the most rostroventral part of the primary somatosensory cortex (S1) and the border between the ventral part of the secondary somatosensory cortex (S2) and the insular oral region (IOR). The kinetics of the optical signal, including the latency, amplitude, rise time and decay time, and half duration, in the S1 region for the response with the largest amplitude were comparable to those in the region with the largest response in S2/IOR. Second, we visualized the regions responding to electrical stimulation of the masseter nerve, which activates both motor efferent fibers and somatosensory afferent fibers, including those that transmit nociceptive and proprioceptive information. Masseter nerve stimulation initially excited the rostral part of the S2/IOR region, and an adjacent region responded to jaw opening. The caudal part of the region showing the maximum response overlapped with the region responding to jaw opening, whereas the rostral part overlapped with the region responding to electrical stimulation of the maxillary and mandibular molar pulps. These findings suggest that proprioception of the masseter is processed in S1 and S2/IOR. Other sensory information, such as nociception, is processed in a region that is adjacent to these pulpal regions and is located in the rostral part of S2/IOR, which receives nociceptive inputs from the molar pulps. The spatial proximity of these regions may be associated with the mechanisms by which masseter muscle pain is incorrectly perceived as dental pain.

本研究では、幼稚園教育実習における学びについて、学生の自己評価の内容を分析することにより、今後の幼稚園教育実習指導の教育課題を検討することを目的として、保育者養成校の大学短期大学部学生の幼稚園教育実習における自己評価をテキストマイニング手法により分析を行った。計量テキスト分析により、幼稚園教育実習中の学生の様子がわかり、学生の評価すべき事項、今後の課題となる事項など明らかにすることができた。クラスター分析の結果、「指導計画」、「子どもとのかかわり」、「幼稚園教育実習に対する取り組む姿勢」などの特徴が析出され、7つのクラスターに分類することができた。

O Ensino e a Aprendizagem de Línguas Estrangeiras Modernas sempre cumpriram relevante papel nas sociedades. No mundo moderno, tecnológico e globalizado em que vivemos, saber diferentes línguas se tornou imprescindível tanto do ponto de vista cultural quanto com relação ao mercado de trabalho. Esse fato, atrelado ao intenso processo de internacionalização do conhecimento presente na sociedade moderna, principalmente em contextos acadêmicos, promove a necessidade de dar maior visibilidade ao ensino de línguas, à formação de professores e à produção científica decorrente desses aspectos. O principal objetivo da busca de maior visibilidade é o de compartilhar saberes produzidos em tais contextos, promovendo e ampliando a capacitação de professores e aprendizes para atuarem num cenário de diversidade cultural.

Summary: Altered DNA methylation plays an important role in the onset and progression of kidney disease. However, little is known about how the changes arise in disease states. Here, we report that KAT5-mediated DNA damage repair is essential for the maintenance of kidney podocytes and is associated with DNA methylation status. Podocyte-specific KAT5-knockout mice develop severe albuminuria with increased DNA double-strand breaks (DSBs), increased DNA methylation of the nephrin promoter region, and decreased nephrin expression. Podocyte KAT5 expression is decreased, whereas DNA DSBs and DNA methylation are increased in diabetic nephropathy; moreover, KAT5 restoration by gene transfer attenuates albuminuria. Furthermore, KAT5 decreases DNA DSBs and DNA methylation at the same nephrin promoter region, which indicates that KAT5-mediated DNA repair may be related to DNA methylation status. These results suggest a concept in which an environment of DNA damage repair, which occurs with decreased KAT5, may affect DNA methylation status. : Hishikawa et al. reveal that KAT5-mediated DNA repair is essential for podocyte maintenance and is related to changes in DNA methylation status. Decreased podocyte KAT5 expression may contribute to the pathophysiology of diabetic nephropathy, suggesting a therapeutic target. Keywords: podocyte, DNA damage repair, DNA methylation, diabetic nephropathy

Background In addition to thymic stromal lymphopoietin and IL-33, IL-25 is known to induce T H 2 cytokine production by various cell types, including T H 2 cells, T H 9 cells, invariant natural killer T cells, and group 2 innate lymphoid cells, involved in T H 2-type immune responses. Because both T H 2-type and T H 17-type cells/cytokines are crucial for contact hypersensitivity (CHS), IL-25 can contribute to this by enhancing T H 2-type immune responses. However, the precise role of IL-25 in the pathogenesis of fluorescein isothiocyanate–induced CHS is poorly understood. Objective We investigated the contribution of IL-25 to CHS using Il25 −/− mice. Methods CHS was evaluated by means of measurement of ear skin thickness in mice after fluorescein isothiocyanate painting. Skin dendritic cell (DC) migration, hapten-specific T H cell differentiation, and detection of IL-1β–producing cells were determined by using flow cytometry, ELISA, and immunohistochemistry, respectively. Results In contrast to thymic stromal lymphopoietin, we found that IL-25 was not essential for skin DC migration or hapten-specific T H cell differentiation in the sensitization phase of CHS. Unexpectedly, mast cell– and non–immune cell–derived IL-25 was important for hapten-specific T H 17 cell–mediated rather than T H 2 cell–mediated inflammation in the elicitation phase of CHS by enhancing T H 17-related, but not T H 2-related, cytokines in the skin. In particular, IL-1β produced by dermal DCs in response to IL-25 was crucial for hapten-specific T H 17 cell activation, contributing to induction of local inflammation in the elicitation phase of CHS. Conclusion Our results identify a novel IL-25 inflammatory pathway involved in induction of T H 17 cell–mediated, but not T H 2 cell–mediated, CHS. IL-25 neutralization can be a potential approach for treatment of CHS.