2-Cl-C.OXT-A stimulates contraction through the suppression of phosphodiesterase activity in human induced pluripotent stem cell-derived cardiac organoids.

BACKGROUND: 2-Cl-C.OXT-A (COA-Cl) is a novel synthesized adenosine analog that activates Sphingosine-1-phosphate 1 receptor (S1P1R) and combines with the adenosine A1 receptor (A1R) in G proteins and was shown to enhance angiogenesis and improve the brain function in rat stroke models. However, the role of COA-Cl in hearts remains unclear. COA-Cl, which has a similar structure to xanthine derivatives, has the potential to suppress phosphodiesterase (PDE), which is an important factor involved in the beating of heart muscle. METHODS AND RESULTS: Cardiac organoids with fibroblasts, human induced pluripotent stem cell-derived cardiac myocytes (hiPSC-CMs), and hiPSC-derived endothelial cells (hiPSC-ECs) were cultured until they started beating. The beating and contraction of organoids were observed before and after the application of COA-Cl. COA-Cl significantly increased the beating rate and fractional area change in organoids. To elucidate the mechanism underlying these effects of COA-Cl on cardiac myocytes, pure hiPSC-CM spheroids were evaluated in the presence/absence of Suramin (antagonist of A1R). The effects of COA-Cl, SEW2871 (direct stimulator of S1P1R), two positive inotropes (Isoproterenol [ISO] and Forskolin [FSK]), and negative inotrope (Propranolol [PRP]) on spheroids were assessed based on the beating rates and cAMP levels. COA-Cl stimulated the beating rates about 1.5-fold compared with ISO and FSK, while PRP suppressed the beating rate. However, no marked changes were observed with SEW2871. COA-Cl, ISO, and FSK increased the cAMP level. In contrast, the level of cAMP did not change with PRP or SEW2871 treatment. The results were the same in the presence of Suramin as absence. Furthermore, an enzyme analysis showed that COA-Cl suppressed the PDE activity by half. CONCLUSIONS: COA-Cl, which has neovascularization effects, suppressed PDE and increased the contraction of cardiac organoids, independent of S1P1R and A1R. These findings suggest that COA-Cl may be useful as an inotropic agent for promoting angiogenesis in the future.

The addition of human iPS cell-derived neural progenitors changes the contraction of human iPS cell-derived cardiac spheroids.

BACKGROUND: We havebeen attempting to use cardiac spheroids to construct three-dimensional contractilestructures for failed hearts. Recent studies have reported that neuralprogenitors (NPs) play significant roles in heart regeneration. However, theeffect of NPs on the cardiac spheroid has not yet been elucidated. OBJECTIVE: This studyaims to demonstrate the influence of NPs on the function of cardiac spheroids. METHODS: Thespheroids were constructed on a low-attachment-well plate by mixing humaninduced pluripotent stem (hiPS) cell-derived cardiomyocytes and hiPScell-derived NPs (hiPS-NPs). The ratio of hiPS-NPs was set at 0%, 10%, 20%,30%, and 40% of the total cell number of spheroids, which was 2500. The motionwas recorded, and the fractional shortening and the contraction velocity weremeasured. RESULTS: Spheroidswere formed within 48 h after mixing the cells, except for the spheroidscontaining 0% hiPS-NPs. Observation at day 7 revealed significant differencesin the fractional shortening (analysis of variance; p = 0.01). The bestfractional shortening was observed with the spheroids containing 30% hiPS-NPs.Neuronal cells were detected morphologically within the spheroids under aconfocal microscope. CONCLUSION: Theaddition of hiPS-NPs influenced the contractile function of the cardiacspheroids. Further studies are warranted to elucidate the underlying mechanism.

Magnetoelectric effect in nanogranular FeCo-MgF films at GHz frequencies.

The magnetoelectric effect is a key issue for material science and is particularly significant in the high frequency band, where it is indispensable in industrial applications. Here, we present for the first time, a study of the high frequency tunneling magneto-dielectric (TMD) effect in nanogranular FeCo-MgF films, consisting of nanometer-sized magnetic FeCo granules dispersed in an MgF insulator matrix. Dielectric relaxation and the TMD effect are confirmed at frequencies over 10 MHz. The frequency dependence of dielectric relaxation is described by the Debye-Fröhlich model, taking relaxation time dispersion into account, which reflects variations in the nature of the microstructure, such as granule size, and the inter-spacing between the granules that affect the dielectric response. The TMD effect reaches a maximum at a frequency that is equivalent to the inverse of the relaxation time. The frequency where the peak TMD effect is observed varies between 12 MHz and 220 MHz, depending on the concentration of magnetic metal in the nanogranular films. The inter-spacing of the films decreases with increasing magnetic metal concentration, in accordance with the relaxation time. These results indicate that dielectric relaxation is controlled by changing the nanostructure, using the deposition conditions. A prospective application of these nanogranular films is in tunable impedance devices for next-generation mobile communication systems, at frequencies over 1 GHz, where capacitance is controlled using the applied magnetic field.

Theory of fast nondeterministic physical random-bit generation with chaotic lasers.

We theoretically show that completely stochastic fast physical random bit generation at a rate of more than one gigabit per second can be realized by using lasers with optical delayed feedback which creates high-dimensional chaos of laser light outputs. The theory is based on the mixing property of chaos, which transduces microscopic quantum noise of spontaneous emission in lasers into random transitions between discrete macroscopic states.

Estimation of entropy rate in a fast physical random-bit generator using a chaotic semiconductor laser with intrinsic noise.

We analyze the time for growth of bit entropy when generating nondeterministic bits using a chaotic semiconductor laser model. The mechanism for generating nondeterministic bits is modeled as a 1-bit sampling of the intensity of light output. Microscopic noise results in an ensemble of trajectories whose bit entropy increases with time. The time for the growth of bit entropy, called the memory time, depends on both noise strength and laser dynamics. It is shown that the average memory time decreases logarithmically with increase in noise strength. It is argued that the ratio of change in average memory time with change in logarithm of noise strength can be used to estimate the intrinsic dynamical entropy rate for this method of random bit generation. It is also shown that in this model the entropy rate corresponds to the maximum Lyapunov exponent.

Noise amplification by chaotic dynamics in a delayed feedback laser system and its application to nondeterministic random bit generation.

We present an experimental method for directly observing the amplification of microscopic intrinsic noise in a high-dimensional chaotic laser system, a laser with delayed feedback. In the experiment, the chaotic laser system is repeatedly switched from a stable lasing state to a chaotic state, and the time evolution of an ensemble of chaotic states starting from the same initial state is measured. It is experimentally demonstrated that intrinsic noises amplified by the chaotic dynamics are transformed into macroscopic fluctuating signals, and the probability density of the output light intensity actually converges to a natural invariant probability density in a strongly chaotic regime. Moreover, with the experimental method, we discuss the application of the chaotic laser systems to physical random bit generators. It is experimentally shown that the convergence to the invariant density plays an important role in nondeterministic random bit generation, which could be desirable for future ultimate secure communication systems.

Ly49Q ligand expressed by activated B cells induces plasmacytoid DC maturation.

Ly49Q, a type II C-type lectin expressed on mouse plasmacytoid DC (pDC), contains a single carbohydrate recognition domain in its extracellular region and an ITIM in its cytoplasmic domain. We have identified the MHC class I molecule H-2K(b) as a Ly49Q ligand, confirming prior reports. Although H-2K(b) is expressed on essentially all hematopoietic cells, we found that only CpG-stimulated B cells were able to activate Ly49Q. This discovery correlated with our finding that although H-2K(b) forms clusters on CpG-activated B cells, it is diffusely expressed on resting B cells. Furthermore, CpG-stimulated, but not resting, B cells up-regulated co-stimulatory molecules on pDC. This finding was confirmed by the fact that binding by anti-Ly49Q mAb to Ly49Q led to pDC maturation in vitro. Our results suggest that clustered H-2K(b) on activated B cells act as ligands for Ly49Q and induce pDC maturation in vitro.

The role of CD11b in phagocytosis and dendritic cell development.

Activation of resting T cells is highly dependent on dendritic cells (DCs), which take up antigens and present antigenic peptides to T cells in the context of the major histocompatibility complex (MHC). In this study, we generated a monoclonal antibody, which we call 1C4 that recognizes integrin alpha(M)beta(2) (CD11b/CD18) on the surface of conventional DCs (cDCs) and is internalized after binding. Addition of 1C4 inhibited the ability of immature DCs to phagocytose apoptotic cells. 1C4 treatment also partially inhibited the generation of cDCs from bone marrow in the presence of granulocyte macrophage colony-stimulating factor (GM-CSF). Our findings suggest that not only CD11b is involved in the phagocytosis of apoptotic cells, but also that mAb such as 1C4 may be a useful tool for the delivery of specific proteins into the cytoplasm of immature DCs.

SAGE library screening reveals ILT7 as a specific plasmacytoid dendritic cell marker that regulates type I IFN production.

Plasmacytoid dendritic cells (pDCs) link innate to acquired immune responses by producing high levels of type I IFN upon infection. In order to identify the specific genes that control pDC, we compared serial analysis of gene expression libraries from human pDCs, herpes simplex virus-stimulated pDCs and monocytes. We found that Ig-like transcript ILT7 is specifically expressed on pDC cell surfaces and is down-regulated when pDC mature in response to viral or bacterial stimulation. ILT7 expression on the cell surface required association with the Fc epsilon RI gamma adaptor molecule. Although treatment with one anti-ILT7-specific mAb suppressed type I IFN production in response to cytosine-phosphate-guanosice (CpG) stimulation, another anti-ILT7 mAb up-regulated type I IFN production. We conclude that ILT7 is a key regulator of human pDC function.

Type I interferon regulates pDC maturation and Ly49Q expression.

Ly49Q is expressed on peripheral mouse plasmacytoid dendritic cells (pDC). Immature Ly49Q-negative pDC precursors acquire Ly49Q in the bone marrow and then migrate into the periphery. While searching for molecules that regulate pDC maturation, we found that type I interferon (IFN) inhibited Ly49Q acquisition in vitro. Infections that induce type I IFN production by cells other than pDC (a condition mimicked by poly(I:C) injection in vivo) increase the prevalence of Ly49Q(-) pDC in the bone marrow and peripheral lymphoid organs in wild-type but not IFN-alpha/beta receptor knockout BALB/c mice. Moreover, in vivo exposure to type I IFN causes some Ly49Q(-), but not Ly49Q(+), pDC to convert to conventional DC, defined as B220(-) CD11c(+) CD11b(+) cells. These data suggest that type I IFN regulates pDC development and affects their distribution in the body.

B cell potential can be obtained from pre-circulatory yolk sac, but with low frequency.

The ontogenic source of definitive hematopoietic system has been identified in non-mammalian vertebrates such as birds and amphibians by orthotopic embryo grafting, but remains unclear for mammals because of technical difficulties. Here, we successfully generated mouse chimeras by grafting yolk sac (YS) on YS of the host embryos before establishing circulation between YS and embryo proper and cultured the whole embryo for 66 h. Donor YS were isolated from C57BL/6 Ly-5.1 and EGFP-transgenic mouse embryos, and recipient embryos from C57BL/6 Ly-5.2 mouse. Almost one-half of the grafts in YS-YS chimeras survived and had obvious blood flow; graft-derived cells comprised 12.7+/-0.9% of the blood cells in the circulation. These graft-derived blood cells consisted mainly of erythroid cells, some myeloid cells and a few blastic cells. In addition, CD19(+) B cells were generated from the graft-derived cells isolated from aorta-gonad-mesonephros (AGM) regions of the YS-YS chimeras; however, the frequency of the YS-derived B cell was low (1.0+/-0.6%) when co-cultured with OP9 stromal cells. These results demonstrate that B cell potential exists in YS before the circulation. Although the major source for B cell is intra-embryonic AGM region, YS may contribute to definitive lymphopoiesis in vivo in mice.

Phosphorylation of MCM4 by Cdc7 kinase facilitates its interaction with Cdc45 on the chromatin.

Cdc7 kinase, conserved from yeasts to human, plays important roles in DNA replication. However, the mechanisms by which it stimulates initiation of DNA replication remain largely unclear. We have analyzed phosphorylation of MCM subunits during cell cycle by examining mobility shift on SDS-PAGE. MCM4 on the chromatin undergoes specific phosphorylation during S phase. Cdc7 phosphorylates MCM4 in the MCM complexes as well as the MCM4 N-terminal polypeptide. Experiments with phospho-amino acid-specific antibodies indicate that the S phase-specific mobility shift is due to the phosphorylation at specific N-terminal (S/T)(S/T)P residues of the MCM4 protein. These specific phosphorylation events are not observed in mouse ES cells deficient in Cdc7 or are reduced in the cells treated with siRNA specific to Cdc7, suggesting that they are mediated by Cdc7 kinase. The N-terminal phosphorylation of MCM4 stimulates association of Cdc45 with the chromatin, suggesting that it may be an important phosphorylation event by Cdc7 for activation of replication origins. Deletion of the N-terminal non-conserved 150 amino acids of MCM4 results in growth inhibition, and addition of amino acids carrying putative Cdc7 target sequences partially restores the growth. Furthermore, combination of MCM4 N-terminal deletion with alanine substitution and deletion of the N-terminal segments of MCM2 and MCM6, respectively, which contain clusters of serine/threonine and are also likely targets of Cdc7, led to an apparent nonviable phenotype. These results are consistent with the notion that the N-terminal phosphorylation of MCM2, MCM4, and MCM6 may play functionally redundant but essential roles in initiation of DNA replication.

Transcriptional repressor foxl1 regulates central nervous system development by suppressing shh expression in zebra fish.

We identified zebra fish forkhead transcription factor l1 (zfoxl1) as a gene strongly expressed in neural tissues such as midbrain, hindbrain, and the otic vesicle at the early embryonic stage. Loss of the function of zfoxl1 effected by morpholino antisense oligonucleotide resulted in defects in midbrain and eye development, and in that of formation of the pectoral fins. Interestingly, ectopic expression of shh in the midbrain and elevated pax2a expression in the optic stalk were observed in foxl1 MO-injected embryos. In contrast, expression of pax6a, which is negatively regulated by shh, was suppressed in the thalamus and pretectum regions, supporting the idea of augmentation of the shh signaling pathway by suppression of foxl1. Expression of zfoxl1-EnR (repressing) rather than zfoxl1-VP16 (activating) resulted in a phenotype similar to that induced by foxl1-mRNA, suggesting that foxl1 may act as a transcriptional repressor of shh in zebra fish embryos. Supporting this notion, foxl1 suppressed isolated 2.7-kb shh promoter activity in PC12 cells, and the minimal region of foxl1 required for its transcriptional repressor activity showed strong homology with the groucho binding motif, which is found in genes encoding various homeodomain proteins. In view of all of our data taken together, we propose zfoxl1 to be a novel regulator of neural development that acts by suppressing shh expression.

Zebrafish Numb homologue: phylogenetic evolution and involvement in regulation of left-right asymmetry.

Numb and its homologue, Numb-like (Numbl), play important roles in mammalian development, but their role in embryonic development of lower vertebrates remains unknown. We cloned a zebrafish numb homologue (znumb) by searching database. znumb shows approximately 60% identity with mammalian Numb orthologs. Interstingly, znumb lacks two specific sequence motifs unique to Numbl orthologs. However, chromosomal localization of znumb gene revealed colinearity with genes located around mouse and human Numbl genes. Furthermore, multi-species comparisons of conserved phosphotyrosine-binding (PTB) domain sequences in Numb and Numbl proteins suggest that znumb is more closely related to Numbl than Numb. znumb mRNA was expressed in a wide variety of zebrafish adult tissues. Overexpression of znumb in embryos resulted in an absence, or reversal, of the normal leftward shift of the developing heart tube. Furthermore, no or bi-lateral transcripts of lefty2 were observed in znumb-expressing embryos, suggesting that the Notch signaling was essential for left-right field formation and maintenance in zebrafish, and that znumb perturbed this process through down-regulation of endogenous Notch signaling.

Hsk1 kinase is required for induction of meiotic dsDNA breaks without involving checkpoint kinases in fission yeast.

Cdc7 kinase, conserved through evolution, is known to be essential for mitotic DNA replication. The role of Cdc7 in meiotic recombination was suggested in Saccharomyces cerevisiae, but its precise role has not been addressed. Here, we report that Hsk1, the Cdc7-related kinase in Schizosaccharomyces pombe, plays a crucial role during meiosis. In a hsk1 temperature-sensitive strain (hsk1-89), meiosis is arrested with one nucleus state before meiosis I in most of the cells and meiotic recombination frequency is reduced by one order of magnitude, whereas premeiotic DNA replication is delayed but is apparently completed. Strikingly, formation of meiotic dsDNA breaks (DSBs) are largely impaired in the mutant, and Hsk1 kinase activity is essential for these processes. Deletion of all three checkpoint kinases, namely Cds1, Chk1, and Mek1, does not restore DSB formation, meiosis, or Cdc2 activation, which is suppressed in hsk1-89, suggesting that these aberrations are not caused by known checkpoint pathways but that Hsk1 may regulate DSB formation and meiosis. Whereas transcriptional induction of some rec genes and horsetail movement are normal, chromatin remodeling at ade6-M26, a recombination hotspot, which is prerequisite for subsequent DSB formation at this locus, is not observed in hsk1-89. These results indicate unique and essential roles of Hsk1 kinase in the initiation of meiotic recombination and meiosis.

The orphan nuclear receptor GCNF recruits DNA methyltransferase for Oct-3/4 silencing.

Somatic DNA methylation patterns are determined in part by the de novo methylation that occurs after early embryonic demethylation. Oct-3/4, a pluripotency gene, is unmethylated in the blastocyst, but undergoes de novo methylation and silencing during gastrulation. Here we show that the transcriptional repressor GCNF recruits DNA methyltransferase to the Oct-3/4 promoter and facilitates its methylation. Although acetylation of histone H3 at lysine 9 (K9) and/or 14 (K14) and methylation of H3 at lysine 4 (K4) decrease during this period, as do Oct-3/4 transcript levels, H3K9 and H3K27 methylation levels remain constant, indicating that DNA methylation does not require repressive histone modifications. We found that GCNF interacts directly with Dnmt3 molecule(s) and verified that this interaction induces the methylation of the Oct-3/4 promoter. Our finding suggests a model in which differentiation-induced GCNF recruits de novo DNA methyltransferase and facilitates the silencing of a pluripotency gene.

SSEA-1 marks regionally restricted immature subpopulations of embryonic retinal progenitor cells that are regulated by the Wnt signaling pathway.

Identification and expansion of retinal progenitor cells are critical issues from both scientific and clinical aspects. Here, we identified SSEA-1 (CD15) as a novel surface antigen that can be used to define immature retinal progenitor cells. SSEA-1-expressing retinal cells were found in the peripheral region of the early embryonic mouse retina, and then their number dramatically disappeared along with retinal development. FACS analysis showed that the cells strongly positive for SSEA-1 co-expressed Ki67 proliferation antigen in all the developmental stages examined. The SSEA-1-expressing cells formed larger colonies than the non-expressing ones in retinal re-aggregation cultures. Moreover, late onset of rhodopsin expression was observed in SSEA-1-positive progenitor cells, supporting the idea that these cells have an intrinsically immature character. Differential expression of Wnt signal-related genes between SSEA-1-positive and -negative subpopulations of retina cells was revealed, and the expression of constitutively active forms of Wnt signaling molecules resulted in a greater number of SSEA-1-positive cells. In light of all of the data taken together, we propose SSEA-1 to be a surface marker to define a regionally restricted immature subset of progenitor cells of mouse neural retina, with SSEA-1 expression by them positively regulated by Wnt signals.