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AbstractOnline social networking services involve communication activities between large number of individuals over the public Internet and their crawled records are often regarded as proxies of real (i.e., offline) interaction structure. However, structure observed in these records might differ from real counterparts because individuals may behave differently online and non-human accounts may even participate. To understand the difference between online and real social networks, we investigate an empirical communication network between users on Twitter, which is perhaps one of the largest social networking services. We define a network of user pairs that send reciprocal messages. Based on the correlation between degree of adjacent nodes observed in this network, we hypothesize that this network differs from conventional understandings in the sense that there is a small number of distinctive users that we call outsiders. Outsiders do not belong to any user groups but they are connected with different groups, while not being well connected with each other. We identify outsiders by maximizing the degree assortativity coefficient of the network via node removal, thereby confirming that local structural properties of outsiders identified are consistent with our hypothesis. Our findings suggest that the existence of outsiders should be considered when using Twitter communication networks for social network analysis.

We investigated whether mesenchymal stem cell (MSC)-based treatment could inhibit neointimal hyperplasia in a rat model of carotid arterial injury and explored potential mechanisms underlying the positive effects of MSC therapy on vascular remodeling/repair. Sprague-Dawley rats underwent balloon injury to their right carotid arteries. After 2 days, we administered cultured MSCs from bone marrow of GFP-transgenic rats (0.8 × 106cells, n = 10) or vehicle (controls, n = 10) to adventitial sites of the injured arteries. As an additional control, some rats received a higher dose of MSCs by systemic infusion (3 × 106cells, tail vein; n = 4). Local vascular MSC administration significantly prevented neointimal hyperplasia (intima/media ratio) and reduced the percentage of Ki67 + proliferating cells in arterial walls by 14 days after treatment, despite little evidence of long-term MSC engraftment. Notably, systemic MSC infusion did not alter neointimal formation. By immunohistochemistry, compared with neointimal cells of controls, cells in MSC-treated arteries expressed reduced levels of embryonic myosin heavy chain and RM-4, an inflammatory cell marker. In the presence of platelet-derived growth factor (PDGF-BB), conditioned medium from MSCs increased p27 protein levels and significantly attenuated VSMC proliferation in culture. Furthermore, MSC-conditioned medium suppressed the expression of inflammatory cytokines and RM-4 in PDGF-BB-treated VSMCs. Thus, perivascular administration of MSCs may improve restenosis after vascular injury through paracrine effects that modulate VSMC inflammatory phenotype.

BackgroundNevoid basal cell carcinoma syndrome (NBCCS) is an autosomal dominant disorder characterised by developmental defects and tumorigenesis, such as medulloblastomas and basal cell carcinomas, caused by mutations of the patched-1(PTCH1) gene. In this article, we seek to demonstrate a mosaicism containing double mutations in PTCH1in an individual with NBCCS.Methods and resultsA de novo germline mutation of PTCH1(c.272delG) was detected in a 31-year-old woman with NBCCS. Gene analysis of two out of four induced pluripotent stem cell (iPSC) clones established from the patient unexpectedly revealed an additional mutation, c.274delT. Deep sequencing confirmed a low-prevalence somatic mutation (5.5%–15.6% depending on the tissue) identical to the one found in iPSC clones.ConclusionsThis is the first case of mosaicism unequivocally demonstrated in NBCCS. Furthermore, the mosaicism is unique in that the patient carries one normal and two mutant alleles. Because these mutations are located in close proximity, reversion error is likely to be involved in this event rather than a spontaneous mutation. In addition, this study indicates that gene analysis of iPSC clones can contribute to the detection of mosaicism containing a minor population carrying a second mutation.

Structure of networks constructed from mentioning relationships between posts in online media may be valuable for understanding how information and opinions spread in these media. We crawled Twitter to collect tweets and replies to construct a large number of so-called reply trees, each of which was rooted at a tweet and joined by replies. Consistent with the previous literature, we found that the empirical trees were characterized by some long path-like reply trees, large star-like trees, and long irregular trees, although their frequencies were not high. We tested several branching process models to explain the empirical frequency of these types of reply trees as well as more basic quantities such as the distributions of the size and depth of the reply tree. Based on our modeling results, we suggest that the in-degree of the tweet that initiates a reply tree (i.e., the number of times that the tweet is directly mentioned by other reply posts) may play an important role in forming the global shape of the reply tree.

Primordial germ cells (PGCs) are embryonic germ cell precursors. Specification of PGCs occurs under the influence of mesodermal induction signaling during in vivogastrulation. Although bone morphogenetic proteins and Wnt signaling play pivotal roles in both mesodermal and PGC specification, the signal regulating PGC specification remains unknown. Coculture of mouse embryonic stem cells (ESCs) with OP9 feeder cells induces mesodermal differentiation in vitro. Using this mesodermal differentiation system, we demonstrated that PGC‐like cells were efficiently induced from mouse ESCs by extracellular signal‐regulated kinase (ERK) signaling inhibition. Inhibition of ERK signaling by a MAPK/ERK kinase (MEK) inhibitor upregulated germ cell marker genes but downregulated mesodermal genes. In addition, the PGC‐like cells showed downregulation of DNA methylation and formed pluripotent stem cell colonies upon treatment with retinoic acid. These results show that inhibition of ERK signaling suppresses mesodermal differentiation but activates germline differentiation program in this mesodermal differentiation system. Our findings provide a new insight into the signaling networks regulating PGC specification. StemCells2014;32:2668–2678

The use of human induced pluripotent stem (iPS) cells would be of great value for a variety of applications involving drug development studies. Several reports have been published on the differentiation of human iPS cells into hepatocyte-like cells; however, the cells were insufficient for application in drug metabolism studies. In this study, we aimed to establish effective methods for differentiation of human iPS cells into hepatocytes. Two human iPS cell lines were differentiated by addition of activin A, dimethyl sulfoxide, hepatocyte growth factor, oncostatin M, and dexamethasone. The differentiated cells expressed hepatocyte markers and drug-metabolizing enzymes, revealing that the human iPS cells were differentiated into hepatocyte-like cells. Expression of CYP3A4 and UGT1A1 mRNAs increased with treatment with typical inducers of the enzymes, and the response of the cells against the inducers was similar to that of human hepatocytes. Furthermore, the drug-metabolizing activity of CYP3A4, as monitored by testosterone 6/S-hydroxylase activity, was elevated by these inducers. In conclusion, we established methods for differentiation of hepatocyte-like cells expressing drug metabolizing activity from human iPS cells. The hepatocyte-like cells derived from human iPS cells will be useful for drug metabolism studies.

This study demonstrates that podocalyxin, a heavily glycosylated type 1 transmembrane protein, is a glycoprotein ligand of rBC2LCN on human induced pluripotent stem (iPS) cells and embryonic stem (ES) cells. When analyzed by DNA microarray, podocalyxin was found to be highly expressed in both iPS cells and ES cells. The carbohydrate antigens of rBC2LCN are expressed on O‐glycans of podocalyxin, since alkaline hydrolysis greatly reduced the binding of rBC2LCN to human iPS cells and ES cells. rBC2LCN bound to an O‐glycan carrying H type 3 epitope structure isolated from iPS cells, suggesting that H type 3 is a novel pluripotency glycan marker. In comprehensive glycome analysis with a high‐density lectin microarray, we have previously shown that the recombinant N‐terminal domain of the lectin BC2L‐C from Burkholderia cenocepacia(rBC2LCN) binds exclusively to undifferentiated human induced pluripotent stem (iPS) cells and embryonic stem (ES) cells but not to differentiated somatic cells. Here we demonstrate that podocalyxin, a heavily glycosylated type 1 transmembrane protein, is a glycoprotein ligand of rBC2LCN on human iPS cells and ES cells. When analyzed by DNA microarray, podocalyxin was found to be highly expressed in both iPS cells and ES cells. Western and lectin blotting revealed that rBC2LCN binds to podocalyxin with a high molecular weight of more than 240 kDa in undifferentiated iPS cells of six different origins and four ES cell lines, but no binding was observed in either differentiated mouse feeder cells or somatic cells. The specific binding of rBC2LCN to podocalyxin prepared from a large set of iPS cells (138 types) and ES cells (15 types) was also confirmed using a high‐throughput antibody‐overlay lectin microarray. Alkaline digestion greatly reduced the binding of rBC2LCN to podocalyxin, indicating that the major glycan ligands of rBC2LCN are presented on O‐glycans. Furthermore, rBC2LCN was found to exhibit significant affinity to a branched O‐glycan comprising an H type 3 structure (Ka, 2.5 × 104M−1) prepared from human 201B7 iPS cells, indicating that H type 3 is a most probable potential pluripotency marker. We conclude that podocalyxin is a glycoprotein ligand of rBC2LCN on human iPS cells and ES cells.

A combination of only five genes—BCL6, T, c-MYC, MITF, and BAF60C—rapidly and efficiently converts postnatal human amnion into chondrocytes. This direct conversion system from noncartilage tissue to cartilaginous tissue is a major advance toward understanding cartilage development, cell-based therapy, and oncogenesis of chondrocytes.

Abstract. We study approximation of common fixed points of a finite family of nonexpansive mappings and introduce an iterative scheme defined by using a convex combination of mappings. Strong convergence of the iteration is obtained under several types of control conditions.

This paper presents a scalable visualization technique for automatic animation of data-flow visual program execution, and a software architecture to provide a scalable interface for debugging programs, which exploits a multi-focus fisheye viewing algorithm in conjunction with a semantic zooming interface to show various kinds of information at runtime. The architecture also supports users' browsing with the interface by automatically assigning proper focal points, based on information embedded in the debugged programs. Thus, it is possible to provide scalable views and intelligent assistance for browsing dynamically created data-flow networks. We have incorporated these ideas into the visual tracer of the KLIEG visual parallel programming environment.

The jumonji (jmj) gene, obtained by a gene trap strategy, is essential for mouse embryogenesis and is suggested to play important roles in cell growth during development. The amino acid sequence of the Jmj protein includes a nuclear localization signal and a DNA binding motif called the AT-rich interaction domain (ARID). To investigate the biological functions of the Jmj protein, we prepared specific antibodies. Using these antibodies, we showed that the Jmj protein is a 160-kDa protein and localizes in the nuclei of COS-7 cells transfected with jmj cDNA and megakaryocytes from fetal liver which show strong endogenous expression of the jmj gene. Moreover, overexpression of the Jmj protein in COS-7 and NIH3T3 cells remarkably reduced cell proliferation compared with control cells transfected with vector alone. These results show that the Jmj protein acts in cell nuclei and participates in the negative regulation of cell proliferation signaling.

Cells from a patient with a DNA repair-deficiency disorder are anticipated to bear a large number of somatic mutations. Because such mutations occur independently in each cell, there is a high degree of mosaicism in patients' tissues. While major mutations that have been expanded in many cognate cells are readily detected by sequencing, minor ones are overlaid with a large depth of non-mutated alleles and are not detected. However, cell cloning enables us to observe such cryptic mutations as well as major mutations. In the present study, we focused on a fibroblastic cell line that is derived from a patient diagnosed with xeroderma pigmentosum (XP), which is an autosomal recessive disorder caused by a deficiency in nucleotide excision repair. By making a list of somatic mutations, we can expect to see a characteristic pattern of mutations caused by the hereditary disorder. We cloned a cell by generating an iPS cell line and performed a whole-exome sequencing analysis of the progenitor and its iPS cell lines. Unexpectedly, we failed to find causal mutations in the XP-related genes, but we identified many other mutations including homozygous deletion of GSTM1and GSTT1. In addition, we found that the long arm of chromosome 9 formed uniparental disomy in the iPS cell line, which was also confirmed by a structural mutation analysis using a SNP array. Type and number of somatic mutations were different from those observed in XP patients. Taken together, we conclude that the patient might be affected by a different type of the disorder and that some of the mutations that we identified here may be responsible for exhibiting the phenotype. Sequencing and SNP-array data have been submitted to SRA and GEO under accession numbers SRP059858 and GSE55520, respectively.

Abstract Vascular endothelial growth factor (VEGF) has potent endothelial cell mitotic and vascular permeability activity. Several reports have suggested that VEGF may be one of the major factors regulating ascites formation, although no quantitative and systematic analyses have been carried out. To determine the role of VEGF in ascites formation, we examined the expression of VEGF in 13 mouse ascites tumors (5 sarcomas, 3 carcinomas, and 5 hematopoietic malignancies). We found that significant amounts (6-850 ng/mL) of biologically active VEGF accumulated in the ascites fluid of all 13 tumors, particularly in tumors of sarcoma and carcinoma origin (430 ± 234 ng/mL). The microvessel densities in the peritoneal walls of tumor-bearing mice, which are significantly higher than those in healthy mice, basically correlated with but did not parallel VEGF concentrations, suggesting the existence of an additional modulator(s) of the angiogenic process. Administration of anti-mouse VEGF-neutralizing antibody to mice bearing the carcinoma-derived ascites tumor MM2 suppressed ascites accumulation, tumor growth, and tendency to bleed. These results directly demonstrate the crucial role of VEGF in carcinoma-derived ascites tumor formation in vivo.

The following describes the quantitative determining method for phosphatidylcholine (PC) using the HPLC-RI system which we have developed. It uses Lichromsorb, Si 60 (10 μm), 4.6 mm × 250 mm as the column and a mobile phase consisting of n-hexane/isopropanol/water =1:4:1. In this report, we compared data from selected high-purity (60–100 wt%) samples using the HPLC-RI, HPLC-UV and conventional TLC-P methods.

Epidermal growth factor (EGF) induces tyrosine phosphorylation of the Shc adapter protein, which plays an important role in EGF-stimulated mitogenesis. Shc stimulates Ras/mitogen-activated protein kinase (MAPK) through forming a complex with Grb2 at the phosphorylated tyrosine (Y) residue 317. In this study, we identified novel phosphorylation sites of Shc, at Y239 and Y240. To define the Shc pathway further, we used NIH 3T3 cells expressing the previously characterized mutant EGF receptor (EGF-R) which lacks all known autophosphorylation sites but retains EGF-stimulated mitogenesis with selective phosphorylation of Shc. We constructed wild-type (WT) or mutant Shc cDNAs in which Y317 or/and Y239 and Y240 are replaced with phenylalanine (F) and introduced them into NIH 3T3 cells expressing WT or mutant EGF-R. In the WT EGF-R-expressing cells, the Y239/240/317F Shc, but not Y317F or Y239/240F Shc, decreased EGF-stimulated cell growth. In the mutant EGF-R-expressing cells, Y317F Shc or Y239/240F Shc decreased EGF-stimulated cell growth significantly, though Y317F was a little more potent than Y239/240F. Although cells expressing the Y317F Shc hardly activated MAPK in response to EGF, cells expressing the Y239/240F Shc fully activated MAPK. In contrast, Y239/240F Shc, but not Y317F Shc, reduced the EGF-induced c-mycmessage. These results suggest that Shc activates two distinct signaling pathways, Y317 to Ras/MAPK and Y239 and Y240 to another pathway including Myc, and that both are involved in EGF-induced mitogenic signaling.

Previous studies have revealed that the growth inhibition of A431 cells overexpressing epidermal growth factor (EGF) receptors by a high concentration of EGF is mainly due to the expression of cycline dependent kinase (CDK) inhibitor p21(WAF1/Cip1). However, the signal transduction mechanism from the activated EGF receptor to the induction of p21(WAF1/Cip1) gene is still poorly understood. We investigated which signaling pathway plays an important role in p21(WAF1/Cip1) expression and growth inhibition by using specific inhibitors of the signaling molecules. A broad PKC inhibitor, PKCδ inhibitor, but not the conventional PKC inhibitor suppressed the EGF-induced p21(WAF1/Cip1) expression and the growth inhibition of A431 cells. These inhibitors did not alter either the activation of EGF receptor or the stimulation of MAP kinase at detectable levels. Furthermore, we found that the induction of p21(WAF1/Cip1) at the early phase (within 12 hr after stimulation) by a high concentration of EGF was independent of the MAP kinase activation by using dominant negative Ras. These results suggest that PKC, especially PKCδ plays a crucial role in the EGF-induced p21(WAF1/Cip1) expression, resulting in the growth inhibition of A431 cells.

The following describes the quantitative determining method for phosphatidylcholine (PC) using the HPLC-RI system which we have developed. It uses Lichromsorb, Si 60 (10 µm), 4.6 mm × 250 mm as the column and a mobile phase consisting of n-hexane/isopropanol/water =1:4:1. In this report, we compared data from selected high-purity (60–100 wt%) samples using the HPLC-RI, HPLC-UV and conventional TLC-P methods. Under the conditions we described, the HPLC-UV method was somewhat affected by fatty acid compositions. As a result, there were some inconsistencies in the measured values. However, the HPLC-RI method we propose was applicable to PC from both egg yolk and soybeans. In addition, the HPLC-RI method produced data which correlated well with data from the TLC-P method, and this data was highly accurate and exhibited satisfac-tory reproductibility.

1-Aryl-1-propenes were converted into the corresponding 2-arylpropionaldehydes in high yields by treatment with iodine and silver(I)oxide in aqueous dioxane at room temperature.

Introduction:Dilated cardiomyopathy (DCM) is genetic disorders that cause left ventricular dysfunction and heart failure. DCM patients with lamin variant have been reported to show the poor prognosis. Recently we have fabricated human cardiac cell sheets composed of human iPS cell-derived cardiomyocyte and succeeded to develop the contractile force measurement system. The aim of this study is to elucidate the function of cardiac cell sheets using iPS cells derived from DCM patients with lamin variant and discover the mechanisms of DCM.Methods:iPS cells were generated from DCM patients with lamin variants (LMNA p.Q353R, LMNA p.R225X) and puromycin resistance gene under the control of ?-myosin heavy chain promoter was introduced to iPS cells with lenti-virus vector. 201B7 iPS cell line was used as control. After the cardiac differentiation and purification process, cells were cultured onto the temperature responsive culture dishes and monolayered cardiac cell sheets were harvested by lowering temperature. Cardiac cell sheets were transferred to the surface of fibrin gel and the contractile force was measured.Results:The purity of cardiomyocyte was identical among groups (control, 64.5?6.1% (n=6), LMNA p.Q353R, 64.5?7.8% (n=4), LMNA p.R225X, 55.9?4.9% (n=3)). Although beating rate and maximum relaxation velocity were not different among groups, contractile force (control, 1.13?0.34mN (n=11), LMNA p.Q353R, 0.49?0.22mN (n=12), LMNA p.R225X, 0.56?0.18mN (n=7), p<0.01) and maximum contraction velocity (control, 8.32?3.33mN/s (n=11), LMNA p.Q353R, 4.14?1.70mN/s (n=12), LMNA p.R225X, 3.70?1.05mN/s (n=7), p<0.01) were significantly decreased in cardiac tissue with LMNA p.Q353R and LMNA p.R225X compared with that in control, suggesting that some lamin variants might directly affect systolic dysfunction of cardiac tissue. In consistent with the impaired systolic dysfunction, mRNA expression of some contractile proteins including MYL2 and MYH6 was remarkably downregulated in cardiac tissues with LMNA p.Q353R and LMNA p.R225X compared with that in control.Conclusions:Human cardiac tissue with lamin variants might be the promising tissue model for understanding the underlying mechanisms of DCM.