PCR amplification from single DNA molecules on magnetic beads in emulsion: application for high-throughput screening of transcription factor targets

We have developed a novel method of genetic library construction on magnetic microbeads based on solid-phase single-molecule PCR in a fine and robust water-phase compartment formed in water-in-oil (w/o) emulsions. In this method, critically diluted DNA fragments were distributed over the emulsion as templates, where beads crosslinked with multiple primers and other PCR components were encapsulated to form multiple reaction compartments. The delivered DNA was then amplified and covalently immobilized on the beads in parallel, within individual compartments, to construct a genetic library on beads (GLOBE), which was readily applicable to a genomewide global scanning of genetic elements recognized by a defined DNA-binding protein. We constructed a GLOBE of Paracoccus denitrificans and selected gene beads that were bound to the His-tagged transcription factor PhaR by flow cytometry. As a result of flow cytometry screening with an anti-His fluorescent antibody, the PhaR target fragments were enriched 1200-fold from this library with this system. Therefore, this system is a powerful tool for analyzing the transcription network on a genomewide scale

Effect of temperature on achene germination in five Mutisieae understory herbaceous species（Asteraceae）

We measured the final germination rates of achenes, the number of days needed to accomplish 50% of the final germination（T50）, and mortalities under various temperatures in the range 5 to 25℃for five understory peren-nial herbs（four species with large achenes of ca. 10 mg weight : Pertya robusta, P . triloba, P . rigidula and Ain-sliaea acerifolia var. subapoda, and one species with small achenes（1mg）: A. apiculata）to investigate variation of these traits. Achenes were collected in Ogawa and Mito in Ibaraki Prefecture, and in Iwakuni in Fukui Prefec-ture. T50 ranged from 35 to 76 days at 5℃and became shorter with increasing temperature. The final germina-tion rates of most samples were over 95%. Ainsliaea acerifolia had the longest T50（76 days）and the lowest final germination rate（86.1%）at 5℃, showing a dormant ability to some extent. Moreover, A. apiculata possessing small achenes showed a lower final germination rate（83%）at 25℃, and dormancy at higher temperatures. Fun-gal attack was thought to cause the death of achenes during the germination period, and mortalities increased with increasing temperature. However, there were no significant differences in final germination rates and T50s between two populations of P . robusta from Ogawa and Mito, though the Ogawa population had a higher mor-tality than the Mito population（27% vs. 18% at 25℃）. Mortalities were higher in P . triloba and P . rigidula than in the other species. There were intra- and interspecific variations of temperature dependency of achene mortal-ity during germination, and these variations may be one of the factors limiting the distribution of each species

Pressure dependence of the spin dynamics around a quantum critical point : An inelastic neutron scattering study of Ce0.87La0.13Ru2Si2

Inelastic neutron scattering experiments performed on a single crystal of the antiferromagnetic compound Ce$_{0.87}$La$_{0.13}$Ru$_{2}$Si$_{2}$ under applied pressures of up to 5 kbar are reported. A quantum critical point is reached at around 2.6 kbar where long-range magnetic order disappears. The variation of the characteristic energy scales with respect to temperature and pressure is followed and found to saturate in the ordered phase.Comment: 14 pages (6 figures

Pressure-Induced Antiferroquadrupole Order in CeTe

We have discovered that the magnetic phase diagram of CeTe under high pressure becomes quite similar to that of CeB6, strongly suggesting that an antiferroquadrupolar ordering is realized. At 1.2 GPa, the transition temperature increases from 3 K at 0.5 T to 6.3 K at 14.5 T. Since the crystal-field ground state of CeTe is the Γ7 doublet without a quadrupolar degree of freedom, this ordering is considered to be realized through the off-diagonal matrix element between Γ7 and Γ8 excited state, whose energy level is lowered with increasing the pressure.This work was supported by Grants-in-Aid for Scientific Research (No. 21244056 and No. 21102515)

Arthroscopic, histological and MRI analyses of cartilage repair after a minimally invasive method of transplantation of allogeneic synovial mesenchymal stromal cells into cartilage defects in pigs

AbstractBackground aimsTransplantation of synovial mesenchymal stromal cells (MSCs) may induce repair of cartilage defects. We transplanted synovial MSCs into cartilage defects using a simple method and investigated its usefulness and repair process in a pig modelMethodsThe chondrogenic potential of the porcine MSCs was compared in vitro. Cartilage defects were created in both knees of seven pigs, and divided into MSCs treated and non-treated control knees. Synovial MSCs were injected into the defect, and the knee was kept immobilized for 10min before wound closure. To visualize the actual delivery and adhesion of the cells, fluorescence-labeled synovial MSCs from transgenic green fluorescent protein (GFP) pig were injected into the defect in a subgroup of two pigs. In these two animals, the wounds were closed before MSCs were injected and observed for 10min under arthroscopic control. The defects were analyzed sequentially arthroscopically, histologically and by magnetic resonance imaging (MRI) for 3 monthsResultsSynovial MSCs had a higher chondrogenic potential in vitro than the other MSCs examined. Arthroscopic observations showed adhesion of synovial MSCs and membrane formation on the cartilage defects before cartilage repair. Quantification analyses for arthroscopy, histology and MRI revealed a better outcome in the MSC-treated knees than in the non-treated control kneesConclusionsLeaving a synovial MSC suspension in cartilage defects for 10min made it possible for cells to adhere in the defect in a porcine cartilage defect model. The cartilage defect was first covered with membrane, then the cartilage matrix emerged after transplantation of synovial MSCs

Metabolomics reveals inosine 5′-monophosphate is increased during mice adipocyte browning

Adipocyte browning is one of the potential strategies for the prevention of obesity-related metabolic syndromes, but it is a complex process. Although previous studies make it increasingly clear that several transcription factors and enzymes are essential to induce browning, it is unclear what dynamic and metabolic changes occur in induction of browning. Here, we analyzed the effect of a beta-adrenergic receptor agonist (CL316243, accelerator of browning) on metabolic change in mice adipose tissue and plasma using metabolome analysis and speculated that browning is regulated partly by inosine 5'-monophosphate (IMP) metabolism. To test this hypothesis, we investigated whether Ucp-1, a functional marker of browning, mRNA expression is influenced by IMP metabolism using immortalized adipocytes. Our study showed that mycophenolic acid (MPA), an IMP dehydrogenase inhibitor, increases the mRNA expression of Ucp-1 in immortalized adipocytes. Furthermore, we performed a single administration of mycophenolate mofetil (MMF), a prodrug of MPA, to mice and demonstrated that MMF induces adipocyte browning and miniaturization of adipocyte size, leading to adipose tissue weight loss. These findings showed that IMP metabolism has a significant effect on adipocyte browning, suggesting that the regulator of IMP metabolism has the potential to prevent obesity

人工被陰下での光合成特性と実生の生存からみたハルジオンの耐陰性

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