Search

Endothelial cellular stiffening has been observed not only in inflamed cultured endothelial cells but also in the endothelium of atherosclerotic regions, which is an underlying cause of monocyte adhesion and accumulation. Although recombinant soluble thrombomodulin (rsTM) has been reported to suppress the inflammatory response of endothelial cells, its role in regulating endothelial cellular stiffness remains unclear. The purpose of this study was to investigate the impact of anticoagulant rsTM on lipopolysaccharide (LPS)-induced endothelial cellular stiffening. We show that LPS increases endothelial cellular stiffness by using atomic force microscopy and that rsTM reduces LPS-induced cellular stiffening not only through the attenuation of actin fiber and focal adhesion formation but also via the improvement of gap junction functionality. Moreover, post-administration of rsTM, after LPS stimulation, attenuated LPS-induced cellular stiffening. We also found that endothelial cells regulate leukocyte adhesion in a substrate- and cellular stiffness-dependent manner. Our result show that LPS-induced cellular stiffening enhances monocytic THP-1 cell line adhesion, whereas rsTM suppresses THP-1 cell adhesion to inflamed endothelial cells by reducing cellular stiffness. Endothelial cells increase cellular stiffness in reaction to inflammation, thereby promoting monocyte adhesion. Treatment of rsTM reduced LPS-induced cellular stiffening and suppressed monocyte adhesion in a cellular stiffness-dependent manner.

Nonalcoholic fatty liver disease (NAFLD) is characterized by a spectrum of liver pathologies, from simple steatosis to steatohepatitis. Recent studies have increasingly noted the aberrant expression of microRNAs closely related to NAFLD pathologies. We have previously shown the presence of increased levels of microRNA-27b (miR-27b) in patients with NAFLD. In this study, we investigated the role of miR-27b in NAFLD by examining the impact of up-regulated miR-27b on the differentiation of preadipocytes into mature adipocytes. We found that miR-27b-3p remarkably enhances the adipocyte differentiation of 3T3-L1 cells associated with lipid accumulation and intracellular triglyceride contents. Furthermore, we have demonstrated not only that miR-27b-3p induces acyl-CoA thioesterase 2 (ACOT2) expression in 3T3-L1 cells, but also that the knockdown of ACOT2 suppresses lipid accumulation and adipocyte differentiation in both the presence and absence of miR-27b-3p treatment. Our data strongly suggest that the miR-27b-ACOT2 axis is an important pathway in adipocyte differentiation and may play a role in the pathogenesis of NAFLD.

Angiogenesis—the sprouting and growth of new blood vessels from the existing vasculature—is an important contributor to tumor development, since it facilitates the supply of oxygen and nutrients to cancer cells. Endothelial cells are critically affected during the angiogenic process as their proliferation, motility, and morphology are modulated by pro-angiogenic and environmental factors associated with tumor tissues and cancer cells. Recent in vivo and in vitro studies have revealed that the gap junctions of endothelial cells also participate in the promotion of angiogenesis. Pro-angiogenic factors modulate gap junction function and connexin expression in endothelial cells, whereas endothelial connexins are involved in angiogenic tube formation and in the cell migration of endothelial cells. Several mechanisms, including gap junction function-dependent or -independent pathways, have been proposed. In particular, connexins might have the potential to regulate cell mechanics such as cell morphology, cell migration, and cellular stiffness that are dynamically changed during the angiogenic processes. Here, we review the implication for endothelial gap junctions and cellular mechanics in vascular angiogenesis.

Angiogenesis, the sprout and growth of new blood vessels from existing vasculature, is an important process of tumor development for the supply of oxygen and nutrition to cancer cells. Endothelial cell is a critical player in angiogenic process by modulating cell proliferation, cell motility, and cell morphology in the response to pro-angiogenic factors and environments provided by tumor and cancer cells. Recent in vivo and in vitro studies have revealed that gap junction of endothelial cells also participates in the promotion of angiogenesis. Pro-angiogenic factors modulate gap junction function and connexins expression in endothelial cells, whereas endothelial connexins involve in angiogenic tube formation and cell migration of endothelial cells via both gap junction channel function dependent or independent mechanisms. In particular, connexin might have the potential to regulate cell mechanics such as cell morphology, cell migration, and cellular stiffness that are dynamically changed during angiogenic processes. Here, we review the implication for endothelial gap junction and cellular mechanics in vascular angiogenesis.

The secretome offers the prospect of new biomarkers for the diagnosis of disease and a promising approach to drug discovery. However, these proteins are secreted in only minute amounts. We propose to use a nanopore to both transfect single cells with nucleic acids and detect secreted protein.

Capillaries pervade human physiology. The mean intercapillary distance is only about 100 μm in human tissue, which indicates the extent of nutrient diffusion. In engineered tissue the lack of capillaries, along with the associated perfusion, is problematic because it leads to hypoxic stress and necrosis. However, a capillary is not easy to engineer due to its complex cytoarchitecture. Here, it is shown that it is possible to create in vitro, in about 30 min, a tubular microenvironment with an elastic modulus and porosity consistent with human tissue that functionally mimicks a bona fide capillary using “live cell lithography”(LCL) to control the type and position of cells on a composite hydrogel scaffold. Furthermore, it is established that these constructs support the forces associated with blood flow and produce nutrient gradients similar to those measured in vivo. With LCL, capillaries can be constructed with single cell precision—no other method for tissue engineering offers such precision. Since the time required for assembly scales with the number of cells, this method is likely to be adapted first to create minimal functional units of human tissue that constitute organs, consisting of a heterogeneous population of 100–1000 cells, organized hierarchically to express a predictable function.

CTRP3/cartducin, a novel C1q family protein, is expressed in proliferating chondrocytes in the growth plate and has an important role in regulating the growth of both chondrogenic precursors and chondrocytes in vitro. We examined the expression of CTRP3/cartducin mRNA in Meckel's cartilage and in condylar cartilage of the fetal mouse mandible. Based on in situ hybridization studies, CTRP3/cartducin mRNA was not expressed in the anlagen of Meckel's cartilage at embryonic day (E)11.5, but it was strongly expressed in Meckel's cartilage at E14.0, and then reduced in the hypertrophic chondrocytes at E16.0. CTRP3/cartducin mRNA was not expressed in the condylar anlagen at E14.0, but was expressed in the upper part of newly formed condylar cartilage at E15.0. At E16.0, CTRP3/cartducin mRNA was expressed from the polymorphic cell zone to the upper part of the hypertrophic cell zone, but was reduced in the lower part of the hypertrophic cell zone. CTRP3/cartducin-antisense oligodeoxynucleotide (AS-ODN) treatment of Meckel's cartilage and condylar anlagen from E14.0 using an organ culture system indicated that, after 4-day culture, CTRP3/cartducin abrogation induced curvature deformation of Meckel's cartilage with loss of the perichondrium and new cartilage formation. Aggrecan, type I collagen, and tenascin-C were simultaneously immunostained in this newly formed cartilage, indicating possible transformation from the perichondrium into cartilage. Further, addition of recombinant mouse CTRP3/cartducin protein to the organ culture medium with AS-ODN tended to reverse the deformation. These results suggest a novel function for CTRP3/cartducin in maintaining the perichondrium. Moreover, AS-ODN induced a deformation of the shape, loss of the perichondrium/fibrous cell zone, and disorder of the distinct architecture of zones in the mandibular condylar cartilage. Additionally, AS-ODN-treated condylar cartilage showed reduced levels of mRNA expression of aggrecan, collagen types I and X, and reduced BrdU-incorporation. These results suggest that CTRP3/cartducin is not only involved in the proliferation and differentiation of chondrocytes, but also contributes to the regulation of mandibular condylar cartilage.

Growing evidence suggests that physical microenvironments and mechanical stresses, in addition to soluble factors, help direct mesenchymal-stem-cell fate. However, biological responses to a local force in embryonic stem cells remain elusive. Here we show that a local cyclic stress through focal adhesions induced spreading in mouse embryonic stem cells but not in mouse embryonic stem-cell-differentiated cells, which were ten times stiffer. This response was dictated by the cell material property (cell softness), suggesting that a threshold cell deformation is the key setpoint for triggering spreading responses. Traction quantification and pharmacological or shRNA intervention revealed that myosin II contractility, F-actin, Src or cdc42 were essential in the spreading response. The applied stress led to oct3/4 gene downregulation in mES cells. Our findings demonstrate that cell softness dictates cellular sensitivity to force, suggesting that local small forces might have far more important roles in early development of soft embryos than previously appreciated.

Background: The major role of mast cells in wound healing process has not been identified. In this study, we used mast cell-deficient W/WV mice and their congenic control (+/+) mice to examine the role of mast cells in scald wound healing. Methods: The size of the scald wound, thickness of the dermis, collagen deposition, vascularization, number of mast cells and chymase activity were measured before and at 3, 7, 14 and 21 days after inducing scald injury. Results: Although the process of wound closure and re-epithelialization was not markedly different between W/WV mice and +/+ mice, the degree of fibrous proliferation at the wound edge and wound vascularization in the proliferative phase was significantly lower in W/WV mice than in +/+ mice, and no vascular regression in the late remodeling phase was observed in W/WV mice. Mast cells producing chymase, FGF2, TGF-β1 and VEGF were highly accumulated at the edge of scald wound in +/+ mice during the proliferative and remodeling phases at days 14 and 21. Chymase activity in the injured tissues of +/+ mice decreased in the acute phase, but recovered to no-injury level at days 14 and 21. The number of mast cells and chymase activity were very low in the injured tissues of W/WV mice throughout the experiment. Conclusions: Wound healing after skin scald injury was partially impaired in mast cell-deficient mice. Mast cells may contribute to the wound healing process, especially in the proliferative and remodeling phases after scald injury.

Despite the recent identification of the transcriptional regulatory circuitry involving SOX2, NANOG, and OCT-4, the intracellular signaling networks that control pluripotency of human embryonic stem cells (hESCs) remain largely undefined. Here, we demonstrate an essential role for the serine/threonine protein kinase mammalian target of rapamycin (mTOR) in regulating hESC long-term undifferentiated growth. Inhibition of mTOR impairs pluripotency, prevents cell proliferation, and enhances mesoderm and endoderm activities in hESCs. At the molecular level, mTOR integrates signals from extrinsic pluripotency-supporting factors and represses the transcriptional activities of a subset of developmental and growth-inhibitory genes, as revealed by genome-wide microarray analyses. Repression of the developmental genes by mTOR is necessary for the maintenance of hESC pluripotency. These results uncover a novel signaling mechanism by which mTOR controls fate decisions in hESCs. Our findings may contribute to effective strategies for tissue repair and regeneration.

This chapter describes standard techniques to (1) maintain mouse embryonic stem cell culture, (2) deliver transgenes into mouse embryonic stem cells mediated by electroporation, nucleofection, lipofection, and retro/lentiviruses, and (3) assess the pluripotency of mouse embryonic stem cells. The last part of this chapter presents induction of random cell differentiation followed by the alkaline phosphatase and embryoid body formation assays, immunofluorescence microscopy, and the teratoma formation assay.

The secretome of a single living cell contains the totality of its secreted proteins,(1) and therefore can act as a fingerprint by which to identify cell type. Although between 10 % and 20 % of the human genome encodes proteins that are secreted, measuring the secretome from an individual living cell is challenging as the secreted proteins are present in vanishingly small concentrations due to the very large dilutions involved.However, a nanopore is able to detect single proteins through the distinctive blockage profile that develops in the ionic conductance current when a protein passes through the nanopore.(2,3) Using a synthetic nanopore in a silicon membrane we investigate the distinctive blockage patterns, in essence the fingerprint, that arise from a single living cell. The cell is placed in proximity to the nanopore using optical tweezers and held stationary. The ionic conductance current is measured across the nanopore, and translocation events (distinct blockage currents) are observed and measured. When the events are plotted in scatter plots (as dwell time versus average blockage current) the distinct fingerprint of individual cells can be observed. For instance, lymphoma cells (U937) and breast cancer cells (MCF7) produce distinct event patterns that enable them to be distinguished. This shows for the first time cell identification based entirely on the secretome, measured using a simple, non-invasive, non-destructive nanopore.(1) Skalnikova, H.; Motlik, J.; Gadher, S.; Kovarova, H. Proteomics, 2011, 11 691-708.(2) Nelson, E. M.; Kurz, V.; Shim, J.; Timp, W.; Timp, G. Analyst, 2012, 137, 3020.(3) Kurz, V.; Nelson, E. M.; Shim, J.; Timp G. ACS Nano, 2013, 7(5), 4057-4069

This chapter describes standard techniques to (1) maintain mouse embryonic stem cell culture, (2) deliver transgenes into mouse embryonic stem cells mediated by electroporation, nucleofection, lipofection, and retro/lentiviruses, and (3) assess the pluripotency of mouse embryonic stem cells. The last part of this chapter presents induction of random cell differentiation followed by the alkaline phosphatase and embryoid body formation assays, immunofluorescence microscopy, and the teratoma formation assay.

In our earlier attempt to identify genes involved in the maintenance of cellular pluripotency, we found that KH-domain protein Embryonal stem cell-specific gene 1 (Esg1) showed similar expression patterns to those of Oct3/4 (Pou5f1), whereas the forced repression of Oct3/4 in mouse embryonic stem cells immediately downregulated the expression of Esg1. Here we further confirm this overlap by in situ hybridization and immunohistochemical analyses. Both Esg1 transcript and protein exist in the egg and preimplantation embryos. At embryonic day 3.5, blastocyst stage, however, ESG1 protein was more abundant in the inner cell mass (ICM) than in trophectoderm (TE), whereas Esg1 transcript was detected in both the ICM and the TE, particularly in the polar trophectoderm. The presence of an RNA-binding KH-domain in ESG1 led us to search for and identify 902 target transcripts by microarray analysis of immunoprecipitated ESG1 complex. Interaction of 20 target mRNA with ESG1, including Cdc25a, Cdc42, Ezh2, Nfyc and Nr5a2, was further validated by reverse transcriptase-polymerase chain reaction of the immunoprecipitation material, supporting the notion that ESG1 is an RNA-binding protein which associates with specific target transcripts.

As a first step to understand preimplantation development, we performed global gene-expression profiling of morula and blastocyst using the NIA 15k mouse cDNA microarray. Gene expression levels were measured four times for blastocyst and five times for morula. Student’s ttest at the 5% significance level identified 428 genes upregulated and 748 downregulated in blastocyst compared to morula. This trend was consistent with semi-quantative RT-PCR analysis of sample genes. The upregulated genes known to be involved in critical regulatory processes, included Mist1, Id2, Hd1, and Requiem; the downregulated genes included CREB-binding protein, Per3, zinc finger protein 217, Krox-25, and miwi1. Such well-characterized genes and many novel genes provide markers for early stages in development and starting materials for further functional studies. Published by Elsevier Ireland Ltd.

A catalog of mouse genes expressed in early embryos, embryonic and adult stem cells was assembled, including 250000 ESTs, representing approximately 39000 unique transcripts. The cDNA libraries, enriched in full-length clones, were condensed into the NIA 15 and 7.4K clone sets, freely distributed to the research community, providing a standard platform for expression studies using microarrays. They are essential tools for studying mammalian development and stem cell biology, and to provide hints about the differential nature of embryonic and adult stem cells.

Microarray expression profiling of a collection of 15,000 mouse genes with placental and embryonic RNAs revealed candidates for placental-enriched genes, three of which we have confirmed and further characterized. One, Plac1 , strongly expressed in all trophoblast-derived cells in the placenta, has been described earlier (Genomics 68 (2000) 305). Here we report that of the other two, Plac8 expression is restricted to the spongiotrophoblast layer during development, whereas Plac9 is weakly expressed though highly enriched in placenta. For both, cDNAs with complete open reading frames were recovered and exon-intron structures inferred from comparisons of mouse cDNA and genomic sequence. The predicted proteins (112 and 108 amino acids) both contain putative signal peptides, with a coiled-coil segment of mPLAC9 as the only other detected motif. Genomic sequence comparisons reveal that in addition to an apparent pseudogene on chromosome 1, Plac8 is expressed at mouse cytoband 5e3. It is tightly conserved in human in a syntenically equivalent ortholog at 4q21.23. Plac9 is present in a single copy on chromosome 14, with a syntenically equivalent human ortholog at 10q22.3. Putative promoter regions up to 10 kb 5′ of the transcription units for Plac1, Plac8, and Plac9 contain sites for widely-expressed transcription factors which, by analogy to other instances, may be sufficient to explain placental enrichment.

Large-scale gene expression profiling was performed on embryo-derived stem cell lines to identify molecular signatures of pluripotency and lineage specificity. Analysis of pluripotent embryonic stem (ES) cells, extraembryonic-restricted trophoblast stem (TS) cells, and terminally-differentiated mouse embryo fibroblast (MEF) cells identified expression profiles unique to each cell type, as well as genes common only to ES and TS cells. Whereas most of the MEF-specific genes had been characterized previously, the majority (67%) of the ES-specific genes were novel and did not include known differentiated cell markers. Comparison with microarray data from embryonic material demonstrated that ES-specific genes were underrepresented in all stages sampled, whereas TS-specific genes included known placental markers. Investigation of four novel TS-specific genes showed trophoblast-restricted expression in cell lines and in vivo, whereas one uncharacterized ES-specific gene, Esg-1, was found to be exclusively associated with pluripotency. We suggest that pluripotency requires a set of genes not expressed in other cell types, whereas lineage-restricted stem cells, like TS cells, express genes predictive of their differentiated lineage.[Supplemental material is available online at www.genome.org andhttp://lgsun.grc.nia.nih.gov/microarray/data.html]

Mutations in the EDA gene cause anhidrotic ectodermal dysplasia (EDA), with lesions in skin appendage formation. To begin to analyze EDA pathways, we have used expression profiling on 15,000-gene mouse cDNA microarrays, comparing adult mouse skin from wild-type, EDA-defective (Tabby) mice, and Tabby mice supplemented with the EDA-A1 isoform, which is sufficient to rescue multiple Tabby phenotypes. Given the sensitivity of the current microarray system, 8500 genes (60%) were estimated to be expressed, including transcription factors and growth-regulatory genes that had not previously been identified in skin; but only 24 (0.16%), one-third of them novel, showed significant differences between wild type and Tabby. An additional eight genes not included in the 15,000 gene set were shown to have expression differences by real-time RT-PCR. Sixteen of 32 affected genes were restored significantly toward wild-type levels in EDA-A1 transgenic Tabby mice. Significant up-regulation in Tabby skin was observed for several dermal matrix genes, including Col1a1, Col1a2, Col3a1 and SPARC: In contrast, down-regulation occurred for the NEMO/NF-kappa B pathway, already implicated in skin appendage formation, and even more markedly for a second pathway, JNK/c-jun/c-fos and their target genes, that has not previously been clearly associated with skin development. These data are consistent with the regulation of the NF-kappa B pathway by EDA, and support its involvement in the regulation of the JNK pathway as well.

Mammalian inner cell mass cells undergo lineage-specific differentiation into germ layers of endoderm, mesoderm and ectoderm during gastrulation. It has been a long-standing challenge in developmental biology to replicate these organized germ layer patterns in culture. Here we present a method of generating organized germ layers from a single mouse embryonic stem cell cultured in a soft fibrin matrix. Spatial organization of germ layers is regulated by cortical tension of the colony, matrix dimensionality and softness, and cell–cell adhesion. Remarkably, anchorage of the embryoid colony from the 3D matrix to collagen-1-coated 2D substrates of ~1 kPa results in self-organization of all three germ layers: ectoderm on the outside layer, mesoderm in the middle and endoderm at the centre of the colony, reminiscent of generalized gastrulating chordate embryos. These results suggest that mechanical forces via cell–matrix and cell–cell interactions are crucial in spatial organization of germ layers during mammalian gastrulation. This new in vitro method could be used to gain insights on the mechanisms responsible for the regulation of germ layer formation., The three germ layers are formed from the inner cell mass of the mammalian embryo during gastrulation. Here, the authors present a method by which a single mouse embryonic stem cell, derived from inner cell mass, differentiates into the three germ layers in a self-organized manner when cultured in soft fibrin gel.

A search for the dinucleon decay $pp\ensuremath{\rightarrow}{K}^{+}{K}^{+}$ has been performed using $91.6\text{ }\text{ }\mathrm{kton}\ifmmode\cdot\else\textperiodcentered\fi{}\text{yr}$ data from Super-Kamiokande-I. This decay provides a sensitive probe of the $R$-parity-violating parameter ${{\ensuremath{\lambda}}_{112}}^{\ensuremath{'}\ensuremath{'}}$. A boosted decision tree analysis found no signal candidates in the data. The expected background was $0.28\ifmmode\pm\else\textpm\fi{}0.19$ atmospheric neutrino induced events and the estimated signal detection efficiency was $12.6%\ifmmode\pm\else\textpm\fi{}3.2%$. A lower limit of $1.7\ifmmode\times\else\texttimes\fi{}{10}^{32}\text{ }\text{ }\text{years}$ has been placed on the partial lifetime of the decay $^{16}\mathrm{O}\ensuremath{\rightarrow}^{14}\mathrm{C}{K}^{+}{K}^{+}$ at 90% C.L. A corresponding upper limit of $7.8\ifmmode\times\else\texttimes\fi{}{10}^{\ensuremath{-}9}$ has been placed on the parameter ${{\ensuremath{\lambda}}_{112}}^{\ensuremath{'}\ensuremath{'}}$.

We report the development of a single cell gene delivery system based on electroporation using a synthetic nanopore, that is not only highly specific and very efficient but also transfects with single molecule resolution at low voltage (1 V) with minimal perturbation to the cell. Such a system can be used to control gene expression with unprecedented precision--no other method offers such capabilities.

The expression of Xenopus vasa homolog or XVLG1 was examined in oocytes and embryos by whole-mount in situ hybridization and reverse transcription polymerase chain reaction (RT-PCR). To confirm the results in embryos, both methods were also applied to explants of germ plasm-bearing cells (GPBC) from 32-cell embryos and to those of partial embryos deprived of GPBC. By hybridization, XVLG1 ribonucleic acid (RNA) was shown to be present throughout the cytoplasm in oocytes at stages I-III, except for the mitochondrial cloud. It was barely recognizable in a portion of germline cells of embryos at specific stages, notwithstanding that XVLG1 protein was present in those cells almost throughout their life-span. A weak signal for the RNA was detectable in some of the presumptive primordial germ cells (pPGC, descendants of GPBC from the gastrula stage onward) from the late gastrula (stage 12) to the hatching tadpole stage (stage 33/34), and in some of the PGC at stages 49-50. The results for pPGC were confirmed by the hybridization of explants of GPBC at equivalent stages in control embryos. In contrast, XVLG1 RNA was detected in certain somatic cells of embryos until stage 46. These observations were supported in part by the results of RT-PCR for embryos and explants. The possible role of the product of XVLG1 was reconsidered given its presence in both germline and somatic cells.

We examined the transcriptional activity of Oct3/4 (Pou5f1) in mouse embryonic stem cells (mESCs) maintained under standard culture conditions to gain a better understanding of self-renewal in mESCs. First, we built an expression vector in which the Oct3/4 promoter drives the monocistronic transcription of Venus and a puromycin-resistant gene via the foot-and-mouth disease virus self-cleaving peptide T2A. Then, a genetically-engineered mESC line with the stable integration of this vector was isolated and cultured in the presence or absence of puromycin. The cultures were subsequently subjected to Illumina expression microarray analysis. We identified approximately 4600 probes with statistically significant differential expression. The genes involved in nucleic acid synthesis were overrepresented in the probe set associated with mESCs maintained in the presence of puromycin. In contrast, the genes involved in cell differentiation were overrepresented in the probe set associated with mESCs maintained in the absence of puromycin. Therefore, it is suggested with these data that the transcriptional activity of Oct3/4 fluctuates in mESCs and that Oct3/4 plays an essential role in sustaining the basal transcriptional activities required for cell duplication in populations with equal differentiation potential. Heterogeneity in the transcriptional activity of Oct3/4 was dynamic. Interestingly, we found that genes involved in the hedgehog signaling pathway showed unique expression profiles in mESCs and validated this observation by RT-PCR analysis. The expression of Gli2, Ptch1 and Smo was consistently detected in other types of pluripotent stem cells examined in this study. Furthermore, the Gli2 protein was heterogeneously detected in mESC nuclei by immunofluorescence microscopy and this result correlated with the detection of the Oct3/4 protein. Finally, forced activation of Gli2 in mESCs increased their proliferation rate. Collectively, it is suggested with these results that Gli2 may play a novel role in the self-renewal of pluripotent stem cells.

Spatial organization of different epigenomic marks was used to infer functions of the epigenome. It remains unclear what can be learned from the temporal changes of the epigenome. Here, we developed a probabilistic model to cluster genomic sequences based on the similarity of temporal changes of multiple epigenomic marks during a cellular differentiation process. We differentiated mouse embryonic stem (ES) cells into mesendoderm cells. At three time points during this differentiation process, we used high-throughput sequencing to measure seven histone modifications and variants—H3K4me1/2/3, H3K27ac, H3K27me3, H3K36me3, and H2A.Z; two DNA modifications—5-mC and 5-hmC; and transcribed mRNAs and noncoding RNAs (ncRNAs). Genomic sequences were clustered based on the spatiotemporal epigenomic information. These clusters not only clearly distinguished gene bodies, promoters, and enhancers, but also were predictive of bidirectional promoters, miRNA promoters, and piRNAs. This suggests specific epigenomic patterns exist on piRNA genes much earlier than germ cell development. Temporal changes of H3K4me2, unmethylated CpG, and H2A.Z were predictive of 5-hmC changes, suggesting unmethylated CpG and H3K4me2 as potential upstream signals guiding TETs to specific sequences. Several rules on combinatorial epigenomic changes and their effects on mRNA expression and ncRNA expression were derived, including a simple rule governing the relationship between 5-hmC and gene expression levels. A Sox17 enhancer containing a FOXA2 binding site and a Foxa2 enhancer containing a SOX17 binding site were identified, suggesting a positive feedback loop between the two mesendoderm transcription factors. These data illustrate the power of using epigenome dynamics to investigate regulatory functions.

In order to know when the protein of Xenopus vasa homolog (Xenopus vasa-like gene 1, XVLG1) first appears in germ line cells and whether the protein is also present in somatic cells as is vasa protein in Drosophila, the spatio-temporal distribution of the protein in Xenopus embryos was carefully investigated by fluorescent microscopy. Part of the observation was performed by whole-mount immunocytochemistry and immunoblotting. A distinct fluorescence of XVLG1 protein was first recognized in a juxta-nuclear location of germ line cells or presumptive primordial germ cells (pPGC) at stage 12 (late gastrula) and remained associated with the pPGC or primordial germ cells (PGC) throughout the following stages until stage 46 (feeding tadpole). In contrast, weak fluorescence was seen in the animal hemisphere rather than in the vegetal hemisphere of cleaving embryos and in the perinuclear region of somatic cells at stages 10–42 (early gastrula to young tadpole), respectively. Nearly the same pattern as revealed by fluorescence was seen by whole-mount immunocytochemistry, except that a small amount of XVLG1 protein seemed to be present in the germ plasm and pPGC of embryos earlier than stage 12. The presence of the protein in the somatic cells and the PGC was also shown by immunoblotting.

In the process of monoclonal antibody (mAb) production against the 38kDa protein which is lacking in the gastrula arrested mutant embryos in Xenopus we incidentally obtained two kinds of mAb (designated as B11 and 2D10 antibodies, respectively) recognizing tropomyosin (TM) proteins in Xenopus embryos. The characterization of the corresponding antigens to those mAb was performed by immunoblotting and silver staining for two-dimensional (2-D) gels in the present study. The localization of the antigens in Xenopus embryos was also investigated by fluorescent microscopy. By 2-D immunoblots with those mAb, three distinct protein spots or TM isoforms were recognized in Xenopus embryos; a 38 kDa spot with a pl of approximately 4.8 reacted with both antibodies in embryos at stages later than the mid-tailbud (stage 28) and two 30 kDa spots, which are probably isomers, with a pl of approximately 4.8 were detected with 2D10 antibody in embryos at stages extending from the fertilized to the mid-neurula (stage 20). By immunofluorescent microscopy, B11 antibody was shown to react mainly with muscle cells and their precursor cells. In contrast, 2D10 antibody stained the cytoplasm of almost all cells in embryos at stages from the fertilized to the tadpole. Judging from the results obtained with immunoblotting and fluorescent microscopy, it is likely that the 38 kDa spot is a skeletal muscle TM isoform and the two 30 kDa spots are non-muscle TM isoforms.

It is now possible to visualize at nanometer resolution the infection of a living biological cell with virus without compromising cell viability using scanning transmission electron microscopy (STEM). To provide contrast while preserving viability, Escherichia coli and P1 bacteriophages were first positively stained with a very low concentration of uranyl acetate in minimal phosphate medium and then imaged with low-dose STEM in a microfluidic liquid flow cell. Under these conditions, it was established that the median lethal dose of electrons required to kill half the tested population was LD50 = 30 e(-)/nm(2), which coincides with the disruption of a wet biological membrane, according to prior reports. Consistent with the lateral resolution and high-contrast signal-to-noise ratio (SNR) inferred from Monte Carlo simulations, images of the E. coli membrane, flagella, and the bacteriophages were acquired with 5 nm resolution, but the cumulative dose exceeded LD50. On the other hand, with a cumulative dose below LD50 (and lower SNR), it was still possible to visualize the infection of E. coli by P1, showing the insertion of viral DNA within 3 s, with 5 nm resolution.

Searches for a nucleon decay into a charged anti-lepton (e^+ or {\mu}^+) plus a light meson ({\pi}^0, {\pi}^-, {\eta}, {\rho}^0, {\rho}^-, {\omega}) were performed using the Super-Kamiokande I and II data. Twelve nucleon decay modes were searched for. The total exposure is 140.9 kiloton \cdot years, which includes a 91.7 kiloton \cdot year exposure (1489.2 live days) of Super-Kamiokande-I and a 49.2 kiloton \cdot year exposure (798.6 live days) of Super-Kamiokande-II. The number of candidate events in the data was consistent with the atmospheric neutrino background expectation. No significant evidence for a nucleon decay was observed in the data. Thus, lower limits on the nucleon partial lifetime at 90% confidence level were obtained. The limits range from 3.6 \times 10^31 to 8.2 \times 10^33 years, depending on the decay modes., Comment: 25 pages, 18 figures

Rhox6 is one of the Reproductive Homeobox genes on the X chromosome (Rhox) that is expressed in the placenta and the post-migratory primordial germ cells (PGCs) in the nascent gonad. Despite its novel expression pattern, the significance of Rhox6 expression in the differentiation of these cell types remains unknown. To investigate the role that Rhox6 plays in PGCs, cDNA encoding Rhox6 and short-hairpin (sh) RNA directed against Rhox6 transcripts were introduced by unique expression vectors into a genetically engineered mouse embryonic stem cell (ESC) line. This ESC line expresses enhanced green fluorescent protein (EGFP) under the Oct3/4 promoter, thereby allowing us to monitor the presence of undifferentiated ESCs and PGCs in culture in real time. This ESC line was used to isolate clones that stably expressed Rhox6 cDNA, shRNA against Rhox6 transcripts, or controls. Quantitative RT-PCR results validated that overexpression had been achieved, as well as knockdown of Rhox6 transcripts in these ESC clones. However, these clones exhibited a normal appearance of undifferentiated ESCs and expressed EGFP. Next, these ESC clones were induced to differentiate into PGCs by generating embryoid bodies (EBs) in culture medium without leukemia inhibitory factor. Detection of EGFP expression by fluorescence microscopy and germ cell markers by RT-PCR validated the differentiation of PGCs in EBs. The Rhox6 transgene had little, if any, effect on EGFP expression in EBs, whereas Rhox6 knockdown significantly decreased EGFP expression in EBs. Thus, it is suggested with these results that Rhox6 is necessary for determination of the germ cell lineage.

In this paper we study non-standard neutrino interactions as an example of physics beyond the standard model using atmospheric neutrino data collected during the Super-Kamiokande I(1996-2001) and II(2003-2005) periods. We focus on flavor-changing-neutral-currents (FCNC), which allow neutrino flavor transitions via neutral current interactions, and effects which violate lepton non-universality (NU) and give rise to different neutral-current interaction-amplitudes for different neutrino flavors. We obtain a limit on the FCNC coupling parameter, varepsilon_{mu tau}, |varepsilon_{mu tau}, 12 Pages, 14 figures. To be submitted to Phys. Rev. D

Here, we present evidence that the tumor-like growth of mouse embryonic stem cells (mESCs) is suppressed by short-term serum-free culture, which is reversed by pharmacological inhibition of Gsk3β. Mouse ESCs maintained under standard conditions using fetal bovine serum (FBS) were cultured in a uniquely formulated chemically-defined serum-free (CDSF) medium, namely ESF7, for three passages before being subcutaneously transplanted into immunocompromised mice. Surprisingly, the mESCs failed to produce teratomas for up to six months, whereas mESCs maintained under standard conditions generated well-developed teratomas in five weeks. Mouse ESCs cultured under CDSF conditions maintained the expression of Oct3/4, Nanog, Sox2 and SSEA1, and differentiated into germ cells in vivo. In addition, when mESCs were cultured under CDSF conditions supplemented with FBS, or when the cells were cultured under CDSF conditions followed by standard culture conditions, they consistently developed into teratomas. Thus, these results validate that the pluripotency of mESCs was not compromised by CDSF conditions. Mouse ESCs cultured under CDSF conditions proliferated significantly more slowly than mESCs cultured under standard conditions, and were reminiscent of Eras-null mESCs. In fact, their slower proliferation was accompanied by the downregulation of Eras and c-Myc, which regulate the tumor-like growth of mESCs. Remarkably, when mESCs were cultured under CDSF conditions supplemented with a pharmacological inhibitor of Gsk3β, they efficiently proliferated and developed into teratomas without upregulation of Eras and c-Myc, whereas mESCs cultured under standard conditions expressed Eras and c-Myc. Although the role of Gsk3β in the self-renewal of ESCs has been established, it is suggested with these data that Gsk3β governs the tumor-like growth of mESCs by means of a mechanism different from the one to support the pluripotency of ESCs.

Embryonic stem cells (ESCs) are derived from preimplantation embryos and are capable of both long-term proliferation (self-renewal) and differentiation into cell types of all three germ layers (pluripotency). The self-renewal and pluripotency of ESCs are sustained by certain essential transcription factors. Intriguingly, the viral transduction of these transcription factors into differentiated adult somatic cells results in reprogramming of the developmental process that the somatic cells have undergone. Consequently, pluripotent cells similar to ESCs, termed induced pluripotent stem cells, can be artificially established from specialized cells. These two types of pluripotent stem cells (PSCs) have held the promise of providing customized tissue replacements as well as platforms for drug screening since they were derived from human tissues and embryos. However, the heterogeneous nature of PSC cultures, which may reflect the plasticity of early embryonic cells, hampers the establishment of a definitive and reproducible culture microenvironment. In addition, the induction of PSC differentiation is dependent on random events and generates heterogeneous populations of specialized cells. Furthermore, PSCs, by definition, are able to generate benign tumors called teratomas, which consist of cell types of three germ layers. To prevent the growth of teratomas in therapeutic transplanted tissue replacements, it is necessary to establish techniques for efficiently manipulating cell fate decisions in PSCs and to understand the mechanism responsible for tumorigenesis in the stem cells. To our surprise, the mechanism of teratoma formation from PSCs has received little attention to date. Thus, in order to better understand self-renewal, pluripotency and tumorigenesis in PSCs, this chapter will address the following three simple but overlooked questions: 1. Does every pluripotent stem cell possess identical self-renewal capability? 2. Are current standard culture conditions optimal for maintaining pluripotent stem cells? 3. Is tumorigenesis an inherent feature of cellular pluripotency? Accumulating experimental evidence, including our recent studies using mouse ESCs as a model, indicates that the self-renewal of PSCs can be easily compromised by extrinsic factors in the culture microenvironment that can turn the stem cells tumorigenic. Thus, the safety of PSC-based therapy may be significantly improved by more careful manipulation and definition of the cellular microenvironment.

Presumptive primordial germ cells (pPGCs) in explants, derived from single germ plasm-bearing cells of Xenopus 32-cell embryos, at the equivalent of neurula stage (stage 20) in control embryos (designated as ‘stage-20′ explants) were demonstrated to be able to differentiate into PGCs, when implanted into a prospective place of pPGCs in host embryos (stage 20) (Ikenishi & Tsuzaki, 1988). According to a recent proposal that individual early embryonic cells in Xenopus, at both in vivo and in vitro, are able to measure elapsed time since fertilization (Cooke and Smith, 1990), the result means that the implanted pPGCs having the same elapsed time as the host embryos (isochronic pPGCs) could differentiate into PGCs. In the present study, in order to know whether the compatibility in elapsed times of implanted pPGCs and host embryos is necessary for the differentiation of PGCs, labelled, heterochronic pPGCs in ‘stages 12–33/34′ explants were implanted into unlabelled, host neurulae (stage 19). Those heterochronic pPGCs could differentiate into PGCs like isochronic pPGCs in ‘stage-19′ explants as the control. By comparing the average diameters and yolk contents of labelled PGCs with those of unlabelled, host ones in experimental tadpoles, the possibility that a certain mechanism modulating the elapsed time of heterochronic pPGCs to that of host pPGCs is present in host embryos was also suggested.

Interleukin 2 (IL-2) and interferon gamma (IFN-gamma) production by CD4+ T cells and IFN-gamma production by CD8+ T cells from naive mice in response to soluble anti-CD3 and antigen-presenting cells (APCs) were strikingly inhibited by culture in the presence of IL-4. IL-4 decreased IL-2 and IFN-gamma mRNA levels after 15-24 hr but gave relatively little decrease in these mRNAs at 6-12 hr after stimulation with soluble anti-CD3. A 16-hr preculture of T cells with anti-CD3, APCs, and IL-4 was sufficient to inhibit subsequent production of IL-2 and IFN-gamma in response to restimulation in the absence of IL-4. Furthermore, IL-4 treatment of T cells purified 24 hr after stimulation inhibited their capacity to subsequently produce IL-2 in response to anti-CD3 and APCs, indicating that T cells were targets of IL-4-mediated inhibition. IL-4 blocked acute IL-2 production in response to a cytochrome c peptide of T cells derived from transgenic mice expressing T-cell receptors specific for cytochrome c but it did not block IL-2 production by such cells after they had been primed in vitro. Nor did IL-4 inhibit production of IFN-gamma by cloned T cells in response to antigen and APCs or production of IL-2 and IFN-gamma by naive T cells in response to phorbol ester and calcium ionophore. These results indicate that IL-4 strikingly inhibits IL-2 and IFN-gamma production by naive T cells in response to accessory cell-dependent, receptor-mediated stimulation (i.e., soluble anti-CD3 and APCs or antigen and APCs) but does not inhibit accessory cell-independent stimulation of naive T cells or accessory cell-dependent receptor-mediated stimulation of recently primed T cells or cloned T-cell lines.

We have reported elsewhere that offspring from the No. 65 female of Xenopus laevis cleaved normally, but their development was arrested at the onset of gastrulation, like the Ambystoma ova-deficient (o) mutant, irrespective of mating with different wild-type males, and that an acidic, 38 kDa protein present in wild-type eggs was lacking in eggs of the female. In the current study, we first determined the partial amino acid sequence (VANLE) of one of the well-separated tryptic peptides from the protein, which was found in elongation factor 1 delta (Ef1delta) in Xenopus, and finally identified the protein as one of the Ef1delta isoforms, Ef1delta2, by peptide mass spectrometry. RT-PCR analyses for Ef1delta2 and its close homolog Ef1delta1 in wild-type oocytes and embryos demonstrated that both transcripts are maternal and Ef1delta1 is present more abundantly than Ef1delta2 throughout the stages examined. Importantly, the amount of the Ef1delta2 transcript per embryo decreased gradually after gastrulation, in accordance with the gradual decrease of the 38 kDa protein per embryo reported in our earlier study. Because pharmacological inhibition of translation induces gastrulation arrest in wild-type embryos, it is reasonable to conclude that the mutant embryos arrest in development due to the lack of Ef1delta2 that is indispensable for translation. Thus, the present study provides the first molecular information on the cause of the gastrulation-defective mutation in Amphibia.

Combining high-content screening (HCS) with random gene-trap mutagenesis could be a powerful tool to investigate transcriptional networks, cell signaling, chemical genetics, and developmental processes. However, a critical limitation has been poor quantification of reporter expression per cell. To overcome this hurdle, we generated a variety of Gtx-based expression cassettes and re-evaluated translational enhancement of arrayed Gtx segments in tandem by HCS. We then modified the cassette into a new polyA trap vector, which consists of a variant of yellow fluorescent protein, Venus, in combination with the Gtx segments. Expression of Venus was detected in about 60% of trapped genes assayed in embryonic stem cell (ESC) cultures, comparable to expression screening of LacZ-based vectors. Furthermore, tetraploid aggregations using a clone encoding a gene-trap insertion into Twist2 demonstrated identical spatiotemporal expression between Venus and Twist2. This highly sensitive reporter system is amenable to high-throughput expression-based real-time HCS including single cell analyses. genesis 46:347–356, 2008. © 2008 Wiley-Liss, Inc.

SummaryStem cell fate is governed by the integration of intrinsic and extrinsic positive and negative signals upon inherent transcriptional networks. To identify novel embryonic stem cell (ESC) regulators and assemble transcriptional networks controlling ESC fate, we performed temporal expression microarray analyses of ESCs after the initiation of commitment and integrated these data with known genome-wide transcription factor binding. Effects of forced under- or overexpression of predicted novel regulators, defined as differentially expressed genes with potential binding sites for known regulators of pluripotency, demonstrated greater than 90% correspondence with predicted function, as assessed by functional and high-content assays of self-renewal. We next assembled 43 theoretical transcriptional networks in ESCs, 82% (23 out of 28 tested) of which were supported by analysis of genome-wide expression in Oct4 knockdown cells. By using this integrative approach, we have formulated novel networks describing gene repression of key developmental regulators in undifferentiated ESCs and successfully predicted the outcomes of genetic manipulation of these networks.

Interferon regulatory factor-8 (IRF-8)/interferon consensus sequence–binding protein (ICSBP) is a transcription factor that controls myeloid-cell development. Microarray gene expression analysis of Irf-8-/- myeloid progenitor cells expressing an IRF-8/estrogen receptor chimera (which differentiate into macrophages after addition of estradiol) was used to identify 69 genes altered by IRF-8 during early differentiation (62 up-regulated and 7 down-regulated). Among them, 4 lysosomal/endosomal enzyme-related genes (cystatin C, cathepsin C, lysozyme, and prosaposin) did not require de novo protein synthesis for induction, suggesting that they were direct targets of IRF-8. We developed a reporter assay system employing a self-inactivating retrovirus and analyzed the cystatin C and cathepsin C promoters. We found that a unique cis element mediates IRF-8–induced activation of both promoters. Similar elements were also found in other IRF-8 target genes with a consensus sequence (GAAANN[N]GGAA) comprising a core IRF-binding motif and an Ets-binding motif; this sequence is similar but distinct from the previously reported Ets/IRF composite element. Chromatin immunoprecipitation assays demonstrated that IRF-8 and the PU.1 Ets transcription factor bind to this element in vivo. Collectively, these data indicate that IRF-8 stimulates transcription of target genes through a novel cis element to specify macrophage differentiation.