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Single-cell RNA sequencing (scRNA-seq) can determine gene expression in numerous individual cells simultaneously, promoting progress in the biomedical sciences. However, scRNA-seq data are high-dimensional with substantial technical noise, including dropouts. During analysis of scRNA-seq data, such noise engenders a statistical problem known as the curse of dimensionality (COD). Based on high-dimensional statistics, we herein formulate a noise reduction method, RECODE (resolution of the curse of dimensionality), for high-dimensional data with random sampling noise. We show that RECODE consistently resolves COD in relevant scRNA-seq data with unique molecular identifiers. RECODE does not involve dimension reduction and recovers expression values for all genes, including lowly expressed genes, realizing precise delineation of cell fate transitions and identification of rare cells with all gene information. Compared with representative imputation methods, RECODE employs different principles and exhibits superior overall performance in cell-clustering, expression value recovery, and single-cell–level analysis. The RECODE algorithm is parameter-free, data-driven, deterministic, and high-speed, and its applicability can be predicted based on the variance normalization performance. We propose RECODE as a powerful strategy for preprocessing noisy high-dimensional data., 1細胞データ解析の精度が飛躍的に向上する前処理法の開発. 京都大学プレスリリース. 2022-08-09., Clearing the mist hiding the genome. 京都大学プレスリリース. 2022-08-09.

In vitro oogenesis is key to elucidating the mechanism of human female germ-cell development and its anomalies. Accordingly, pluripotent stem cells have been induced into primordial germ cell-like cells and into oogonia with epigenetic reprogramming, yet further reconstitutions remain a challenge. Here, we demonstrate ex vivo reconstitution of fetal oocyte development in both humans and cynomolgus monkeys (Macaca fascicularis). With an optimized culture of fetal ovary reaggregates over three months, human and monkey oogonia enter and complete the first meiotic prophase to differentiate into diplotene oocytes that form primordial follicles, the source for oogenesis in adults. The cytological and transcriptomic progressions of fetal oocyte development in vitro closely recapitulate those in vivo. A comparison of single-cell transcriptomes among humans, monkeys, and mice unravels primate-specific and conserved programs driving fetal oocyte development, the former including a distinct transcriptomic transformation upon oogonia-to-oocyte transition and the latter including two active X chromosomes with little X-chromosome upregulation. Our study provides a critical step forward for realizing human in vitro oogenesis and uncovers salient characteristics of fetal oocyte development in primates., ヒト・サルの胎児卵巣から原始卵胞を体外で作出することに成功. 京都大学プレスリリース. 2022-08-01., New egg recipe to boost fertility research. 京都大学プレスリリース. 2022-08-14.

Germ cells are unique in engendering totipotency, yet the mechanisms underlying this capacity remain elusive. Here, we perform comprehensive and in‐depth nucleome analysis of mouse germ‐cell development in vitro, encompassing pluripotent precursors, primordial germ cells (PGCs) before and after epigenetic reprogramming, and spermatogonia/spermatogonial stem cells (SSCs). Although epigenetic reprogramming, including genome‐wide DNA de‐methylation, creates broadly open chromatin with abundant enhancer‐like signatures, the augmented chromatin insulation safeguards transcriptional fidelity. These insulatory constraints are then erased en masse for spermatogonial development. Notably, despite distinguishing epigenetic programming, including global DNA re‐methylation, the PGCs‐to‐spermatogonia/SSCs development entails further euchromatization. This accompanies substantial erasure of lamina‐associated domains, generating spermatogonia/SSCs with a minimal peripheral attachment of chromatin except for pericentromeres—an architecture conserved in primates. Accordingly, faulty nucleome maturation, including persistent insulation and improper euchromatization, leads to impaired spermatogenic potential. Given that PGCs after epigenetic reprogramming serve as oogenic progenitors as well, our findings elucidate a principle for the nucleome programming that creates gametogenic progenitors in both sexes, defining a basis for nuclear totipotency.

Trophoblast are extra-embryonic cells that are essential to maintain pregnancy. Human trophoblasts arise from the morula as trophectoderm (TE), which, after implantation, differentiates into cytotrophoblast (CT), syncytiotrophoblast (ST), and extravillous trophoblast (EVT) composing the placenta. Here we show that naïve, but not primed, human pluripotent stem cells (PSCs) recapitulate trophoblast development. Naïve PSC-derived TE and CT (nCT) recreated the human and monkey TE-to-CT transition. nCT self-renewed as CT stem cells and had the characteristics of proliferating villous CT and CT in the cell column of the first trimester. Notably, although primed PSCs differentiated into trophoblast-like cells (pBAP), pBAP were distinct from nCT and human placenta-derived CT stem cells, exhibiting properties consistent of the amnion. Our findings establish an authentic paradigm for human trophoblast development, demonstrating the invaluable properties of naïve human PSCs. Our system will provide a platform to study the molecular mechanisms underlying trophoblast development and related diseases.

Trophoblasts are extraembryonic cells that are essential for maintaining pregnancy. Human trophoblasts arise from the morula as trophectoderm (TE), which, after implantation, differentiates into cytotrophoblasts (CTs), syncytiotrophoblasts (STs), and extravillous trophoblasts (EVTs), composing the placenta. Here we show that naive, but not primed, human pluripotent stem cells (PSCs) recapitulate trophoblast development. Naive PSC-derived TE and CTs (nCTs) recreated human and monkey TE-to-CT transition. nCTs self-renewed as CT stem cells and had the characteristics of proliferating villous CTs and CTs in the cell column of the first trimester. Notably, although primed PSCs differentiated into trophoblast-like cells (BMP4, A83-01, and PD173074 [BAP]-treated primed PSCs [pBAPs]), pBAPs were distinct from nCTs and human placenta-derived CT stem cells, exhibiting properties consistent with the amnion. Our findings establish an authentic paradigm for human trophoblast development, demonstrating the invaluable properties of naive human PSCs. Our system provides a platform to study the molecular mechanisms underlying trophoblast development and related diseases.

Reconstituting a human ovary Human pluripotent stem cells (hPSCs) have been induced into human primordial germ cell–like cells (hPGCLCs) in vitro, the first step toward human in vitro gametogenesis. Yamashiro et al. went a step closer to generating mature gametes by culturing hPSCs with mouse embryonic ovarian somatic cells in xenogeneic reconstituted ovaries (see the Perspective by Gill and Peters). Over a period of 4 months, hPGCLCs underwent hallmark epigenetic reprogramming and differentiated progressively into cells closely resembling human oogonia, an immediate embryonic precursor for human oocytes. This study creates opportunities for human germ cell research and provides a foundation for human in vitro gametogenesis. Science , this issue p. 356 ; see also p. 291

Summary Mammalian male germ-cell development consists of three distinct phases: primordial germ cell (PGC) development, male germ-cell specification for spermatogonium development, and ensuing spermatogenesis. Here, we show an in vitro reconstitution of whole male germ-cell development by pluripotent stem cells (PSCs). Mouse embryonic stem cells (mESCs) are induced into PGC-like cells (mPGCLCs), which are expanded for epigenetic reprogramming. In reconstituted testes under an optimized condition, such mPGCLCs differentiate into spermatogonium-like cells with proper developmental transitions, gene expression, and cell-cycle dynamics and are expanded robustly as germline stem cell-like cells (GSCLCs) with an appropriate androgenetic epigenome. Importantly, GSCLCs show vigorous spermatogenesis, not only upon transplantation into testes in vivo but also under an in vitro culture of testis transplants, and the resultant spermatids contribute to fertile offspring. By uniting faithful recapitulations of the three phases of male germ-cell development, our study creates a paradigm for the in vitro male gametogenesis by PSCs.

The human germ-cell lineage originates as human primordial germ cells (hPGCs). hPGCs undergo genome-wide epigenetic reprogramming and differentiate into oogonia or gonocytes, precursors for oocytes or spermatogonia, respectively. Here, we describe a protocol to differentiate human induced pluripotent stem cells (hiPSCs) into oogonia in vitro. hiPSCs are induced into incipient mesoderm-like cells (iMeLCs) using activin A and a WNT pathway agonist. iMeLCs, or, alternatively, hPSCs cultured with divergent signaling inhibitors, are induced into hPGC-like cells (hPGCLCs) in floating aggregates by cytokines including bone morphogenic protein 4. hPGCLCs are aggregated with mouse embryonic ovarian somatic cells to form xenogeneic reconstituted ovaries, which are cultured under an air-liquid interface condition for ~4 months for hPGCLCs to differentiate into oogonia and immediate precursory states for oocytes. To date, this is the only approach that generates oogonia from hPGCLCs. The protocol is suitable for investigating the mechanisms of hPGC specification and epigenetic reprogramming.

This work shows that GATA transcription factors transduce the BMP signaling and, with SOX17 and TFAP2C, induce the human germ-cell fate, delineating the mechanism for human germ-cell specification., The in vitro reconstitution of human germ-cell development provides a robust framework for clarifying key underlying mechanisms. Here, we explored transcription factors (TFs) that engender the germ-cell fate in their pluripotent precursors. Unexpectedly, SOX17, TFAP2C, and BLIMP1, which act under the BMP signaling and are indispensable for human primordial germ-cell-like cell (hPGCLC) specification, failed to induce hPGCLCs. In contrast, GATA3 or GATA2, immediate BMP effectors, combined with SOX17 and TFAP2C, generated hPGCLCs. GATA3/GATA2 knockouts dose-dependently impaired BMP-induced hPGCLC specification, whereas GATA3/GATA2 expression remained unaffected in SOX17, TFAP2C, or BLIMP1 knockouts. In cynomolgus monkeys, a key model for human development, GATA3, SOX17, and TFAP2C were co-expressed exclusively in early PGCs. Crucially, the TF-induced hPGCLCs acquired a hallmark of bona fide hPGCs to undergo epigenetic reprogramming and mature into oogonia/gonocytes in xenogeneic reconstituted ovaries. By uncovering a TF circuitry driving the germ line program, our study provides a paradigm for TF-based human gametogenesis.

Central Nervous System Germ Cell Tumor (CNS GCT) is the second most common pediatric brain tumor in Japan, and within CNS GCT, germinoma is the most common subtype, accounting for 62.3%. Recent reports of transcriptome and methylome analysis suggested that germinoma highly resemble the state of gonocytes, the germ cells at around 5th to 7th week of human embryo development. It is also identified that 60% of germinoma harbored somatic mutations in KIT/RAS pathway. As the protocol to derive gonocytes from human iPSCs have been reported, we aimed to recapitulate tumorigenesis by generating human iPSCs bearing common genetic mutations and derive gonocytes from them. We first introduced the most common mutation KITD816V to human iPSCs using CRISPR/Cas9, and confirmed in iPSCs that mutated KIT was phosphorylated in the absence of ligand stimulation, and also found that KIT activation contribute to the phosphorylation of AKT but not of ERK. Upon differentiation towards primordial germ cell -like cells (PGCLCs), KIT mutant lines were efficiently induced into PGCLCs, however, by comparing conditions with or without KIT ligand (SCF), mutant lines exhibited less dependency to SCF compared to wildtype cells. Mutant cells were further differentiated to gonocytes following published protocol and the cells were collected for transcriptome analysis. By comparing with the transcriptome of germinoma, we confirmed that germinoma samples express germ cell genes similar to gonocytes. We are attempting to identify the molecular mechanism of tumorigenesis in relation to KIT activation using this system.

The timing of developmental events during early mouse development has been investigated in embryos that have been subject to experimental manipulation of cell number and tissue mass. These phenomenological studies revealed that the timing of preimplantation events, such as compaction, formation of blastocyst cavity and lineage allocation is correlated with the rounds of cleavage division or DNA replication of the blastomeres. Timing of postimplantation processes, such as formation of proamniotic cavity and onset of gastrulation is sensitive to cell number and probably the tissue mass, which may be measured by a mechanosensory signaling mechanism. Developmental changes in these two physical attributes are correlated with the cell proliferative activity and the growth trajectory of the whole embryo prior to the transit to organogenesis. During organogenesis, timing of morphogenesis appears to be regulated by individual devices that could be uncoupled during compensatory growth. Insights of the timing mechanism may be gleaned from the analysis of genomic activity associated with the transition through developmental milestones.

SummaryMouse epiblast stem cells (EpiSCs) can be derived from a wide range of developmental stages. To characterize and compare EpiSCs with different origins, we derived a series of EpiSC lines from pregastrula stage to late-bud-stage mouse embryos. We found that the transcriptomes of these cells are hierarchically distinct from those of the embryonic stem cells, induced pluripotent stem cells (iPSCs), and epiblast/ectoderm. The EpiSCs display globally similar gene expression profiles irrespective of the original developmental stage of the source tissue. They are developmentally similar to the ectoderm of the late-gastrula-stage embryo and behave like anterior primitive streak cells when differentiated in vitro and in vivo. The EpiSC lines that we derived can also be categorized based on a correlation between gene expression signature and predisposition to differentiate into particular germ-layer derivatives. Our findings therefore highlight distinct identifying characteristics of EpiSCs and provide a foundation for further examination of EpiSC properties and potential.

Cardiac transcription factors play crucial roles in cardiac development and differentiation. To address the target genes they regulate is important for understanding the molecular mechanisms underlying these processes. In this study, ES cell lines harboring a neomycin resistance gene in the Nkx2-5 gene locus were generated and used to make purified ES cell-derived cardiomyocytes (ESCM) by in vitro differentiation and successive G418 treatment. Three lines with different Nkx2-5 gene expression levels were made and named as Nkx2-5 -/-, +/-, and overexpressing ESCMs. In order to investigate Nkx2-5-regulated gene expression in cardiomyocytes, the gene expression profiles were compared among these lines. The expression patterns of known Nkx2-5 downstream genes were consistent with the previous investigations in vivo. Several genes with Nkx2-5-dependent changes in their expression were detected and confirmed by real-time PCR. This study investigated Nkx2-5-regulated gene expression in ESCM and suggested potential targets of Nkx2-5 in cardiogenesis.

Objective— CCN1 (Cyr61) is an extracellular matrix-associated protein involved in cell proliferation and survival. CCN1 is bound to vascular smooth muscle cells (VSMCs) via integrins and is expressed in VSMCs in atherosclerotic lesions, suggesting involvement in the regulation of vascular smooth muscle cell (VSMC) proliferation and atherosclerosis. We hypothesized that knockdown of CCN1 may inhibit VSMC proliferation and suppress neointimal hyperplasia. Methods and Results— We examined the effect of the knockdown of CCN1 using rat cultured VSMCs and a rat balloon injury model. CCN1 stimulated adhesion and migration of VSMCs in a dose-dependent manner, and this was blocked by an antibody for integrin α 6 β 1 . Moreover, knockdown of endogenous CCN1 by lentiviral delivery of siRNA significantly inhibited proliferation of VSMCs and the uptake of 5-bromo-2′-deoxyuridine (BrdU). Replenishment with recombinant CCN1 reversed the effect of siRNA knockdown. Interestingly, knockdown of CCN1 significantly suppressed neointimal hyperplasia in a rat carotid artery balloon injury model at days 14 and 28 after injury. Gene transfer of CCN1 to smooth muscle reversed the effect of CCN1 knockdown on neointimal formation. These results suggest that endogenous CCN1 regulates proliferation of VSMCs and neointimal hyperplasia. Conclusion— Inhibition of CCN1 may provide a promising strategy for the prevention of restenosis after vascular interventions.

CCN1 (Cyr61) is a secreted matricellular protein, mediating angiogenesis and cell survival through interaction with integrins. Although CCN1 expression is induced in the heart during ischemia and pressure overload, its function in cardiac myocytes remains to be elucidated. We hypothesized that CCN1 may not only induce angiogenesis but may also have a direct effect on cardiac myocytes during ischemia. In this study, we investigated the effect of CCN1 on survival of cardiac myocytes under oxidative stress and examined a signal transduction pathway downstream of CCN1. A solid-phase binding assay demonstrated that CCN1 was bound to cardiac myocytes in a dose-dependent, saturable manner. Inactivation of beta1 integrin in cardiac myocytes inhibited binding with CCN1, indicating that CCN1 was bound to cardiac myocytes via beta1 integrin. Knockdown of endogenous CCN1 decreased the number of surviving cells under oxidative stress, while pretreatment of cardiac myocytes with recombinant CCN1 significantly increased the number of surviving cells. Moreover, TUNEL staining showed that CCN1 significantly decreased apoptotic cells. Furthermore, treatment of cardiac myocytes with CCN1 induced phosphorylation of Akt and extracellular signal-regulated kinase (ERK). Inactivation of beta1 integrin inhibited CCN1-induced phosphorylation of these kinases and abolished the protective effect of CCN1. Moreover, pretreatment of cells with wortmannin completely blocked the protective effect of CCN1 on cardiac myocytes under oxidative stress, indicating that the protective effect of CCN1 was mainly mediated by activation of Akt. The antiapoptotic effect of CCN1 on cardiac myocytes together with its proangiogenic property could be beneficial in the treatment of ischemic heart disease.

A 69-year-old female suffering from third-degree atrioventricular block with syncope underwent permanent pacemaker implantation. However, she developed shortness of breath 2 months after the implantation. Blood tests revealed elevated levels of LDH, CRP, BNP, and SIL-2R. Transthoracic echocardiography showed thickened left and right atrial walls with mild pericardial effusion. A diagnosis was made based on a CT scan and histology. Although most primary cardiac malignant lymphomas are associated with a poor prognosis, the patient was treated successfully with chemotherapy.

Germline specification underlies human reproduction and evolution, but it has proven difficult to study in humans since it occurs shortly after blastocyst implantation. This process can be modeled with human induced pluripotent stem cells (hiPSCs) by differentiating them into primordial germ cell-like cells (hPGCLCs) through an incipient mesoderm-like cell (iMeLC) state. Here, we elucidate the key transcription factors and their interactions with important signaling pathways in driving hPGCLC differentiation from iPSCs. Germline competence of iMeLCs is dictated by the duration and dosage of WNT signaling, which induces expression of EOMES to activate SOX17, a key driver of hPGCLC specification. Upon hPGCLC induction, BMP signaling activates TFAP2C in a SOX17-independent manner. SOX17 and TFAP2C then cooperatively instate an hPGCLC transcriptional program, including BLIMP1 expression. This specification program diverges from its mouse counterpart regarding key transcription factors and their hierarchies, and it provides a foundation for further study of human germ cell development.

We examined the effects of tetragastrin on mucin (mucus glycoprotein) content and mucosal damage in the rat stomach and duodenum. Following an injection of tetragastrin (12, 120, or 400 microg/kg subcutaneously), no macroscopic damage was found to the gastric mucosa but an increase in corpus mucin content was noted, whereas mucosal lesions appeared and the mucin content decreased in the duodenum in a dose-related manner. In the groups with histamine (0.8, 8, or 80 mg/kg intraperitoneally) administration, the extent of mucosal damage and the decrease in mucin content were dose-related in both these regions. For assessment of the effect of tetragastrin on the protective action in gastroduodenal mucosa, changes in mucin content and mucosal damage with histamine (80 mg/kg) -induced injury were examined. Coadministration of tetragastrin prevented the gastric mucosal damage and inhibited the decrease in corpus mucin content. In the duodenum, tetragastrin aggravated the histamine-induced mucosal damage and did not inhibit the reduction of the mucin content. From the present results, the increase in gastric mucins induced by tetragastrin might be related to the protective effect of gastric mucosa against injury. Tetragastrin did not protect the duodenal mucosa, and histamine-induced injury occurring in this region would be aggravated by the increase in HCl secretion and the decrease in mucin content induced by tetragastrin.

WNT signaling activity is involved in the regulation of many cellular functions, including proliferation, migration, cell fate specification, maintenance of pluripotency and induction of tumorigenicity. Here we summarize recent progress towards understanding the regulation of canonical WNT/β-catenin signaling activity through feedback regulatory loops involving the ligands, agonists and antagonists, the availability of intracellular pools of active β-catenin and the cross-regulation of the WNT activity by β-catenin independent pathway. We also review recent findings on the role of WNT/β-catenin signaling in tissue lineage differentiation during embryogenesis and the maintenance and self renewal of embryo-derived stem cells in vitro.

Background— Recently, elevation of circulating muscle-specific microRNA (miRNA) levels has been reported in patients with acute myocardial infarction. However, it is still unclear from which part of the myocardium or under what conditions miRNAs are released into circulating blood. The purpose of this study was to identify the source of elevated levels of circulating miRNAs and their function in cardiovascular diseases. Methods and Results— Serum levels of miRNA (miR)-1 and miR-133a were increased significantly in patients not only with acute myocardial infarction but also with unstable angina pectoris and Takotsubo cardiomyopathy without elevation of serum creatine phosphokinase or cardiac troponin. MicroRNA microarray analysis of the heart from a mouse model of myocardial infarction indicated that the levels of miR-1, miR-133a, miR-208a, and miR-499 were significantly reduced in the infarcted myocardium. In situ hybridization of miR-133a also showed that miR-133a levels were very low in the infarcted and peri-infarcted myocardium. It has been shown that circulating miRNAs are localized inside exosomes, which are released after Ca 2+ stimulation. We stimulated H9c2 cardiomyoblasts with A23187 and measured miR-133a levels in the exosome fraction of the culture medium. A23187 induced a dose-dependent release of miR-133a, and significant elevation was observed only at concentrations where dead cells were detected. We also found that miR-133a–containing exosomes reduced the luciferase activity of 293FT cells transfected with an miR-133a sensor vector. Conclusions— These results suggest that elevated levels of circulating miR-133a in patients with cardiovascular diseases originate mainly from the injured myocardium. Circulating miR-133a can be used as a marker for cardiomyocyte death, and it may have functions in cardiovascular diseases.

Changes in gastric mucus glycoprotein (mucin) isolated from pirenzepine-treated rats with or without ethanol (50%)-induced gastric damage were studied. The prior administration of pirenzepine inhibited significantly and dose-dependently the occurrence of macroscopically observable hemorrhagic lesions induced by treatment with ethanol. The gastric mucosa was separated into the surface mucosa, including the mucus gel layer, and the deeper mucosa by mechanical scraping, and the mucin in each was isolated. In cthanol-treated animals the mucin content of the deep corpus mucosa was significantly reduced to 68% the control value. This reduction was inhibited by pretreatment with pirenzepine. In the surface mucosa mucin content was also reduced to 48% of control value by ethanol treatment, but pirenzepine pretreatment increased mucin content to 235% the control value. Total mucin content in the entire stomach essentially resumed the control level by pretreatment with 100 mg/kg of pirenzepine. A single oral admini...

Screening for cell surface proteins up-regulated under stress conditions may lead to the identification of new therapeutic targets. To search for genes whose expression was enhanced by treatment with oligomycin, a mitochondrial-F0F1 ATP synthase inhibitor, signal sequence trapping was performed in H9C2 rat cardiac myoblasts. One of the genes identified was that for neural cell adhesion molecule (NCAM, CD56), a major regulator of development, cell survival, migration, and neurite outgrowth in the nervous system. Immunohistochemical analyses in a mouse myocardial infarction model revealed that NCAM was strongly expressed in residual cardiac myocytes in the infarcted region. Increased expression of NCAM was also found during the remodeling period in a rat model of hypertension-induced heart failure. Lentivirus-mediated knockdown of NCAM decreased the cell growth and survival following oligomycin treatment in H9C2 cells. In primary rat neonatal cardiac myocytes, NCAM was also found to be up-regulated and played a protective role following oligomycin treatment. Analyses of downstream signaling revealed that knockdown of NCAM significantly decreased the basal AKT phosphorylation level. In contrast, NCAM mimetic peptide P2d activated AKT and significantly reduced oligomycin-induced cardiomyocyte death, which was abolished by treatment with the PI3K inhibitor LY-294002 as well as overexpression of the dominant-negative AKT mutant. These findings demonstrate that NCAM is a cardioprotective factor up-regulated under metabolic stress in cardiomyocytes and augmentation of this signal improved survival.

Mouse epiblast stem cells (EpiSCs) display temporal differences in the upregulation ofMixl1expression during the initial steps ofin vitrodifferentiation, which can be correlated with their propensity for endoderm differentiation. EpiSCs that upregulatedMixl1rapidly during differentiation responded robustly to both Activin A and Nodal in generating foregut endoderm and precursors of pancreatic and hepatic tissues. By contrast, EpiSCs that delayed Mixl1 upregulation responded less effectively to Nodal and showed an overall suboptimal outcome of directed differentiation. The enhancement in endoderm potency in Mixl1-early cells may be accounted for by a rapid exit from the progenitor state and the efficient response to the induction of differentiation by Nodal. EpiSCs that readily differentiate into the endoderm cells are marked by a distinctive expression fingerprint of transforming growth factor (TGF)-β signalling pathway genes and genes related to the endoderm lineage. Nodal appears to elicit responses that are associated with transition to a mesenchymal phenotype, whereas Activin A promotes gene expression associated with maintenance of an epithelial phenotype. We postulate that the formation of definitive endoderm (DE) in embryoid bodies follows a similar process to germ layer formation from the epiblast, requiring an initial de-epithelialization event and subsequent re-epithelialization. Our results show that priming EpiSCs with the appropriate form of TGF-β signalling at the formative phase of endoderm differentiation impacts on the further progression into mature DE-derived lineages, and that this is influenced by the initial characteristics of the cell population. Our study also highlights that Activin A, which is commonly used as anin vitrosurrogate for Nodal in differentiation protocols, does not elicit the same downstream effects as Nodal, and therefore may not effectively mimic events that take place in the mouse embryo.

The effects of the muscarinic agonist carbachol on the secretion and accumulation of gastric mucus glycoprotein (mucin) were examined. Gastric mucin obtained from the soluble mucus, adherent mucus gel, and surface mucosal and deep mucosal layer was isolated and quantified. One hour after the subcutaneous administration of carbachol (0.08-80 micrograms/kg body weight) the deep corpus mucin content had decreased significantly (75% of control), corresponding to an increase (120% of control) in the soluble mucin content with 0.8 microgram/kg of carbachol treatment. These changes were counteracted by 10 mg/kg of pirenzepine (selective M1 antagonist) pretreatment. On a single administration of 10 mg/kg of pirenzepine, deep corpus mucin tended to increase, and soluble mucin decreased significantly (49% of the control). These two drugs failed to cause any significant change in the mucin content of the surface and antral deep mucosa or the adherent mucus gel. The muscarinic agonist and the M1 antagonist are thus shown to accelerate the secretion and accumulation, respectively, of mucin in the deep corpus mucosa. Thus intrinsic M1 receptor may possibly be involved in the secretion of mucin in the gastric deep corpus mucosa.

合成清酒の濃味は清酒より多少劣り, その一因がアミノ酸の不足によることは明らかである。そこで, 大豆蛋白質, アルブミン, ゼラチンの3種の蛋白質を配合した蛋白質混合物を完全加水分解し, 生成したアミノ酸を分離・精製し, 清酒様アミノ酸組成を有する合成清酒用のアミノ酸調味料を製造することを検討した。その結果, 得られたアミノ酸混合物の収率は65%で, ほとんど無色・無臭の粉末であり, 数種のアミノ酸の多少の違いを除けば清酒中のアミノ酸組成に近似していることがペーパー・クロマトグラフィーおよびアミノ酸自動分析機によるアミノ酸分析から確められた。次いで, このアミノ酸混合物の調味料としての合成清酒への効果を官能および緩衝能の面から調べると, 呈味的には合成清酒の濃味を増して酒質を改善し, 緩衝能の面ではpH8.0～10.0の緩衝能を高め合成清酒の緩衝能を清酒の緩衝能に近づける効果があることがわかった。以上の結果から, この製造法で作られるアミノ酸混合物は合成清酒の酒質の改善に有効な調味料であることを確めた。

Although a number of papers about the structure theory or the control theory of sliver have been published by many authers, it seems difficult to say that both of them have enough supported to each other. One of main reasons might depend on great difficulties to determine the distribution of fiber ends in sliver which is considered as the definite representation of sliver structure. In this report some trials to make the structure theory relate with the control theory are shown using a model sliver which consists of fibers having even thickness and length.At the first part of this report, a way of determining the positions of fiber ends in sliver from the sliver thickness which can be easily measured is discussed. Considering the relation between the fiber end positions and the sliver thickness, it is shown that positions of fiber ends can not be detected from the sliver thickness except those which are located at intervals of fiber length. Therefore it is clearly impossible to remove the irregularity within the fiber length only with the imformation of sliver thickness. Then a new method is proposed. This is, measuring and comparing the sliver thickness before and after drafting, positions of fiber ends at intervals of 1/N times fiber length can be detected if drafting ratio used is expressed by a rational number N/M (>1), where M and N are integers. In this way, it becomes possible to control the very short range irregularity of sliver thickness theoretically. This method also makes posible to count up the number of fiber ends existing in each intervals which divide the sliver along the length.In this paper, the length L/N, where L is the whole length of sliver and N is the total number of fibers, is chosen as the elementary interval and the standard deviation which is calculated from the set of the number of fiber ends contained in each elementary interval is assumed as a representative index for the irregularity of sliver thickness. However the elementary interval is usually so narrow that n times one would be reasonably used for practical observation. Then the next problems are how to estimate the standard deviation for the elementary interval from data for the n times elementary interval and to predict the irregularity of sliver thickness after control which is performed with data obtained by the n times elementary interval. For this purpose a method based on the maximum entropy principle of imformation theory is proposed and, applying this method to a random sliver, the expected irregularity of thickness controlled by ideal draft mechanism is investigated. Main results are as follows1. The expected irregularity of thickness is less improved when the sliver is controlled with data obtained by larger interval. This tendency rapidly becomes dull and, by and by, sliver control based on data for a interval over the critical one becomes ineffective.2. When the draft ratio smaller than one is forbidden to use, the expected irregularity of thickness after control is less improved than the one in the case of unlimited draft ratio.But results for both case close to each other no matter draft ratio is limited or not, when draftratio is large enough.

清酒および合成清酒について成分の差違を緩衝能を指標として検討した。すなわち, それらのアルカリ滴定曲線を仮に酸区分 (pH 4.0～6.8), 無機塩区分 (pH 6.8～8.0), 高分子区分 (pH 8.0～9.4), アミノ酸区分 (pH 9.4～10.3) および糖区分 (pH 10.3～11.0) に分別し, 各区分の占める割合いを検討した。その結果, 合成清酒の緩衝能は酸区分のみが強く, 糖区分, 特に無機塩区分が少ないことを認めた。この事実は清酒と非常に異なっていた。そこで, この点を改良するために, 純合合成清酒の各成分 (酸, 塩類など) の緩衝能を検討した。その結果, 酸類および無機塩類を増すこと, 特にアンモニウム塩を添加することによって, 清酒の滴定曲線とほとんど近似した滴定曲線を示す純合合成清酒の仕込配合を見出した。この仕込配合は酸類および糖類はもとの仕込配合より約10%増加しているし, 特に無機塩類は約3.6倍になっていた。成分的には濃くなっているにも拘わらず, 官能審査では味は薄く収れん性も減じているように認められた。従って, この組成を基礎にして検討を進めれば, 呈味のバランスをくずさず, エキス分を多くし, 良好な合成清酒を造ることができるとの可能性を得たo

A 53-year-old woman who was on hemodialysis underwent the resection of an aneurysm of the descending thoracic aorta as well as graft replacement. The operation was performed with the aid of femoro-femoral bypass perfusion and an artificial kidney inserted on the venous side of bypass line. Although the perfusion time was short, the uremic metabolic substance was efficiently removed, resulting in easy management after surgery.