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1.  Hemogenic endocardium contributes to transient definitive hematopoiesis 
Nature communications  2013;4:1564.
Hematopoietic cells arise from spatiotemporally restricted domains in the developing embryo. Although studies of non-mammalian animal and in vitro embryonic stem cell models suggest a close relationship among cardiac, endocardial, and hematopoietic lineages, it remains unknown whether the mammalian heart tube serves as a hemogenic organ akin to the dorsal aorta. Here we examine the hemogenic activity of the developing endocardium. Mouse heart explants generate myeloid and erythroid colonies in the absence of circulation. Hemogenic activity arises from a subset of endocardial cells in the outflow cushion and atria earlier than in the aorta-gonad-mesonephros region, and is transient and definitive in nature. Interestingly, key cardiac transcription factors, Nkx2-5 and Isl1, are expressed in and required for the hemogenic population of the endocardium. Together, these data suggest that a subset of endocardial/endothelial cells expressing cardiac markers serve as a de novo source for transient definitive hematopoietic progenitors.
doi:10.1038/ncomms2569
PMCID: PMC3612528  PMID: 23463007
2.  Complement receptor C5aR1/CD88 and dipeptidyl peptidase-4/CD26 define distinct hematopoietic lineages of dendritic cells 
Differential display of the integrins CD103 and CD11b are widely used to distinguish two major dendritic cell (DC) subsets in nonlymphoid tissues. CD103+ DCs arise from fms-like tyrosine kinase receptor 3 (FLT3)-dependent DC precursors (preDC), whereas CD11bhi DCs can arise either from preDCs or FLT3-independent monocytes. Functional characterization of these two lineages of CD11bhi DCs has been hindered by the lack of a widely applicable method to distinguish between them. We performed gene expression analysis of fractionated lung DCs from C57BL/6 mice, and found that monocyte-derived (mo)DCs including CD11bhiLy-6Clo tissue resident and CD11bhiLy-6Chi inflammatory moDCs express the complement 5a receptor 1 (C5aR1)/CD88, whereas preDC-derived conventional DCs (cDCs) including CD103+ and CD11bhi cDCs express dipeptidyl peptidase-4/CD26. Flow cytometric analysis of multiple organs, including the kidney, liver, lung, lymph nodes, small intestine and spleen, confirmed that reciprocal display of CD88 and CD26 can reliably distinguish FLT3-independent moDCs from FLT3-dependent cDCs in C57BL/6 mice. Similar results were obtained when DCs from BALB/c mice were analyzed. Using this novel approach to study DCs in mediastinal lymph nodes, we observed that the majority of blood-derived LN resident DCs, as well as tissue-derived migratory DCs, are cDCs. Furthermore, cDCs, but not moDCs, stimulated naïve T cell proliferation. We anticipate that the use of antibodies against CD88 and CD26 to distinguish moDCs and cDCs in multiple organs and mouse strains will facilitate studies aimed at assigning specific functions to distinct DC lineages in immune responses.
doi:10.4049/jimmunol.1402195
PMCID: PMC4390500  PMID: 25769922
3.  Nanoparticle-Mediated Targeting of Cyclosporine A Enhances Cardioprotection Against Ischemia-Reperfusion Injury Through Inhibition of Mitochondrial Permeability Transition Pore Opening 
Scientific Reports  2016;6:20467.
Myocardial ischemia-reperfusion (IR) injury limits the therapeutic effects of early reperfusion therapy for acute myocardial infarction (MI), in which mitochondrial permeability transition pore (mPTP) opening plays a critical role. Our aim was to determine whether poly-lactic/glycolic acid (PLGA) nanoparticle-mediated mitochondrial targeting of a molecule that inhibits mPTP opening, cyclosporine A (CsA), enhances CsA-induced cardioprotection. In an in vivo murine IR model, intravenously injected PLGA nanoparticles were located at the IR myocardium mitochondria. Treatment with nanoparticles incorporated with CsA (CsA-NP) at the onset of reperfusion enhanced cardioprotection against IR injury by CsA alone (as indicated by the reduced MI size at a lower CsA concentration) through the inhibition of mPTP opening. Left ventricular remodeling was ameliorated 28 days after IR, but the treatment did not affect inflammatory monocyte recruitment to the IR heart. In cultured rat cardiomyocytes in vitro, mitochondrial PLGA nanoparticle-targeting was observed after the addition of hydrogen peroxide, which represents oxidative stress during IR, and was prevented by CsA. CsA-NP can be developed as an effective mPTP opening inhibitor and may protect organs from IR injury.
doi:10.1038/srep20467
PMCID: PMC4748220  PMID: 26861678
4.  Flow-induced protein kinase A–CREB pathway acts via BMP signaling to promote HSC emergence 
Kim et al. identify a novel shear stress–induced pathway involving protein kinase A, CREB, and bone morphogenetic protein that regulates hematopoietic stem cell generation in the embryonic aorta.
Fluid shear stress promotes the emergence of hematopoietic stem cells (HSCs) in the aorta–gonad–mesonephros (AGM) of the developing mouse embryo. We determined that the AGM is enriched for expression of targets of protein kinase A (PKA)–cAMP response element-binding protein (CREB), a pathway activated by fluid shear stress. By analyzing CREB genomic occupancy from chromatin-immunoprecipitation sequencing (ChIP-seq) data, we identified the bone morphogenetic protein (BMP) pathway as a potential regulator of CREB. By chemical modulation of the PKA–CREB and BMP pathways in isolated AGM VE-cadherin+ cells from mid-gestation embryos, we demonstrate that PKA–CREB regulates hematopoietic engraftment and clonogenicity of hematopoietic progenitors, and is dependent on secreted BMP ligands through the type I BMP receptor. Finally, we observed blunting of this signaling axis using Ncx1-null embryos, which lack a heartbeat and intravascular flow. Collectively, we have identified a novel PKA–CREB–BMP signaling pathway downstream of shear stress that regulates HSC emergence in the AGM via the endothelial-to-hematopoietic transition.
doi:10.1084/jem.20141514
PMCID: PMC4419355  PMID: 25870201
5.  Thermodynamic properties of water molecules in the presence of cosolute depend on DNA structure: a study using grid inhomogeneous solvation theory 
Nucleic Acids Research  2015;43(21):10114-10125.
In conditions that mimic those of the living cell, where various biomolecules and other components are present, DNA strands can adopt many structures in addition to the canonical B-form duplex. Previous studies in the presence of cosolutes that induce molecular crowding showed that thermal stabilities of DNA structures are associated with the properties of the water molecules around the DNAs. To understand how cosolutes, such as ethylene glycol, affect the thermal stability of DNA structures, we investigated the thermodynamic properties of water molecules around a hairpin duplex and a G-quadruplex using grid inhomogeneous solvation theory (GIST) with or without cosolutes. Our analysis indicated that (i) cosolutes increased the free energy of water molecules around DNA by disrupting water–water interactions, (ii) ethylene glycol more effectively disrupted water–water interactions around Watson–Crick base pairs than those around G-quartets or non-paired bases, (iii) due to the negative electrostatic potential there was a thicker hydration shell around G-quartets than around Watson–Crick-paired bases. Our findings suggest that the thermal stability of the hydration shell around DNAs is one factor that affects the thermal stabilities of DNA structures under the crowding conditions.
doi:10.1093/nar/gkv1133
PMCID: PMC4666364  PMID: 26538600
6.  AI-03TARGETING ANGIOGENESIS WITHOUT INCREASING THE STROMAL CELL RESPONSE OR INVASION USING ABT-898, A THROMBOSPONDIN TYPE 1 REPEAT PEPTIDE 
Neuro-Oncology  2014;16(Suppl 5):v1.
Anti-angiogenic therapy is a promising therapeutic approach for highly vascularized tumors including glioblastoma (GBM). Nonetheless, the efficacy of Bevacizumab - a monoclonal antibody to VEGF-A - is limited only to progression-free survival but not overall survival, due at least in part to the induction of a more invasive phenotype. Here, we investigated the anti-tumor effect of a thrombospondin-1&2 mimetic peptide, ABT-898, on intracerebral xenografts derived from primary patient GBM neurospheres in the nude mouse. ABT-898 treatment of xenograft tumors (80 mg/kg/day i.p.) prolonged mouse survival. Unlike Bevacizumab-treated mouse brain tumors, ABT-898-treated tumors did not exhibit a detectable increase in the number of invasive foci or phospho-Met expression, indicating no induction of an invasive phenotype. As stromal cells are thought to promote tumor cell invasion, we quantitated infiltration of Iba1+ activated microglial/macrophages into the tumor. Although Bevacizumab therapy significantly increased the number of recruited Iba1+ cells into the tumors, the number of Iba1+ cells in the ABT-898-treated tumors was not increased as compared to the vehicle-treated xenograft tumors. Supporting this finding, the migration of microglia cells (BV2), was significantly inhibited by ABT898 in a dose-dependent manner, and the inhibition was reversed by CD36-neutralizing antibody. This suggests that recruitment of tumor invasion-promoting microglia/macrophage can be regulated by ABT898 and this regulation is in part CD36-dependent. In addition, treatment with ABT898 significantly reduced vessel density in xenograft tumors. In vitro ABT898 induced apoptosis of brain endothelial cells (ECs) and inhibited tubulomorphogenesis in collagen gels, both in a dose-dependent manner, and both were reversed by treatment with CD36 neutralizing antibody, Collectively, these data suggest that ABT-898 offers the novel advantage over Bevacizumab of decreasing stromal cell recruitment to the tumor and thus should be considered as an alternative anti-angiogenic therapy for GBM.
doi:10.1093/neuonc/nou238.3
PMCID: PMC4217869
7.  A common variant mapping to CACNA1A is associated with susceptibility to Exfoliation syndrome 
Aung, Tin | Ozaki, Mineo | Mizoguchi, Takanori | Allingham, R Rand | Li, Zheng | Haripriya, Aravind | Nakano, Satoko | Uebe, Steffen | Harder, Jeffrey M. | Chan, Anita S.Y. | Lee, Mei Chin | Burdon, Kathryn P. | Astakhov, Yury S. | Abu-Amero, Khaled K. | Zenteno, Juan C. | Nilgün, Yildirim | Zarnowski, Tomasz | Pakravan, Mohammad | Safieh, Leen Abu | Jia, Liyun | Wang, Ya Xing | Williams, Susan | Paoli, Daniela | Schlottmann, Patricio G | Huang, Lulin | Sim, Kar Seng | Foo, Jia Nee | Nakano, Masakazu | Ikeda, Yoko | Kumar, Rajesh S | Ueno, Morio | Manabe, Shin-ichi | Hayashi, Ken | Kazama, Shigeyasu | Ideta, Ryuichi | Mori, Yosai | Miyata, Kazunori | Sugiyama, Kazuhisa | Higashide, Tomomi | Chihara, Etsuo | Inoue, Kenji | Ishiko, Satoshi | Yoshida, Akitoshi | Yanagi, Masahide | Kiuchi, Yoshiaki | Aihara, Makoto | Ohashi, Tsutomu | Sakurai, Toshiya | Sugimoto, Takako | Chuman, Hideki | Matsuda, Fumihiko | Yamashiro, Kenji | Gotoh, Norimoto | Miyake, Masahiro | Astakhov, Sergei Y. | Osman, Essam A. | Al-Obeidan, Saleh A. | Owaidhah, Ohoud | Al-Jasim, Leyla | Al Shahwan, Sami | Fogarty, Rhys A. | Leo, Paul | Yetkin, Yaz | Oğuz, Çilingir | Kanavi, Mozhgan Rezaei | Beni, Afsaneh Naderi | Yazdani, Shahin | Akopov, Evgeny L. | Toh, Kai-Yee | Howell, Gareth R | Orr, Andrew C. | Goh, Yufen | Meah, Wee Yang | Peh, Su Qin | Kosior-Jarecka, Ewa | Lukasik, Urszula | Krumbiegel, Mandy | Vithana, Eranga N | Wong, Tien Yin | Liu, Yutao | Ashley Koch, Allison E. | Challa, Pratap | Rautenbach, Robyn M | Mackey, David A. | Hewitt, Alex W | Mitchell, Paul | Wang, Jie Jin | Ziskind, Ari | Carmichael, Trevor | Ramakrishnan, Rangappa | Narendran, Kalpana | Venkatesh, Rangaraj | Vijayan, Saravanan | Zhao, Peiquan | Chen, Xueyi | Guadarrama-Vallejo, Dalia | Cheng, Ching Yu | Perera, Shamira A | Husain, Rahat | Ho, Su-Ling | Welge-Luessen, Ulrich-Christoph | Mardin, Christian | Schloetzer-Schrehardt, Ursula | Hillmer, Axel M. | Herms, Stefan | Moebus, Susanne | Nöthen, Markus M. | Weisschuh, Nicole | Shetty, Rohit | Ghosh, Arkasubhra | Teo, Yik Ying | Brown, Matthew A | Lischinsky, Ignacio | Crowston, Jonathan G | Coote, Michael | Zhao, Bowen | Sang, Jinghong | Zhang, Nihong | You, Qisheng | Vysochinskaya, Vera | Founti, Panayiota | Chatzikyriakidou, Anthoula | Lambropoulos, Alexandros | Anastasopoulos, Eleftherios | Coleman, Anne L | Wilson, M Roy | Rhee, Douglas J | Kang, Jae Hee | May-Bolchakova, Inna | Heegaard, Steffen | Mori, Kazuhiko | Alward, Wallace L.M. | Jonas, Jost B | Xu, Liang | Liebmann, Jeffrey M | Chowbay, Balram | Schaeffeler, Elke | Schwab, Matthias | Lerner, Fabian | Wang, Ningli | Yang, Zhenglin | Frezzotti, Paolo | Kinoshita, Shigeru | Fingert, John H. | Inatani, Masaru | Tashiro, Kei | Reis, André | Edward, Deepak P. | Pasquale, Louis R. | Kubota, Toshiaki | Wiggs, Janey L. | Pasutto, Francesca | Topouzis, Fotis | Dubina, Michael | Craig, Jamie E. | Yoshimura, Nagahisa | Sundaresan, Periasamy | John, Simon W.M. | Ritch, Robert | Hauser, Michael A | Khor, Chiea-Chuen
Nature genetics  2015;47(4):387-392.
Exfoliation syndrome (XFS) is the commonest recognizable cause of open angle glaucoma world-wide. To better understand the etiology of XFS, we conducted a genome-wide association study (GWAS) on 1,484 patients and 1,188 controls from Japan, and followed up the most significant findings on a further 6,901 patients and 20,727 controls from 17 countries across 6 continents. We discovered a significant association between a new locus (CACNA1A rs4926244) and increased susceptibility to XFS (Odds ratio [OR] = 1.16, P = 3.36 × 10−11). Although overwhelming association at the LOXL1 locus was confirmed, the key SNP marker (LOXL1 rs4886776) demonstrated allelic reversal depending on ethnic grouping (In Japanese: ORA-allele= 9.87, P = 2.13 × 10−217; In non-Japanese: ORA-allele= 0.49, P = 2.35 × 10−31). Our findings represent the first genetic locus outside of LOXL1 which surpasses genome-wide significance for XFS, and provides insight into the biology and pathogenesis of the disease.
doi:10.1038/ng.3226
PMCID: PMC4605818  PMID: 25706626
8.  A New Therapeutic Modality for Acute Myocardial Infarction: Nanoparticle-Mediated Delivery of Pitavastatin Induces Cardioprotection from Ischemia-Reperfusion Injury via Activation of PI3K/Akt Pathway and Anti-Inflammation in a Rat Model 
PLoS ONE  2015;10(7):e0132451.
Aim
There is an unmet need to develop an innovative cardioprotective modality for acute myocardial infarction (AMI), for which the effectiveness of interventional reperfusion therapy is hampered by myocardial ischemia-reperfusion (IR) injury. Pretreatment with statins before ischemia is shown to reduce MI size in animals. However, no benefit was found in animals and patients with AMI when administered at the time of reperfusion, suggesting insufficient drug targeting into the IR myocardium. Here we tested the hypothesis that nanoparticle-mediated targeting of pitavastatin protects the heart from IR injury.
Methods and Results
In a rat IR model, poly(lactic acid/glycolic acid) (PLGA) nanoparticle incorporating FITC accumulated in the IR myocardium through enhanced vascular permeability, and in CD11b-positive leukocytes in the IR myocardium and peripheral blood after intravenous treatment. Intravenous treatment with PLGA nanoparticle containing pitavastatin (Pitavastatin-NP, 1 mg/kg) at reperfusion reduced MI size after 24 hours and ameliorated left ventricular dysfunction 4-week after reperfusion; by contrast, pitavastatin alone (as high as 10 mg/kg) showed no therapeutic effects. The therapeutic effects of Pitavastatin-NP were blunted by a PI3K inhibitor wortmannin, but not by a mitochondrial permeability transition pore inhibitor cyclosporine A. Pitavastatin-NP induced phosphorylation of Akt and GSK3β, and inhibited inflammation and cardiomyocyte apoptosis in the IR myocardium.
Conclusions
Nanoparticle-mediated targeting of pitavastatin induced cardioprotection from IR injury by activation of PI3K/Akt pathway and inhibition of inflammation and cardiomyocyte death in this model. This strategy can be developed as an innovative cardioprotective modality that may advance currently unsatisfactory reperfusion therapy for AMI.
doi:10.1371/journal.pone.0132451
PMCID: PMC4500569  PMID: 26167913
9.  Trif-dependent induction of Th17 immunity by lung dendritic cells 
Mucosal immunology  2014;8(1):186-197.
Allergic asthma is thought to stem largely from maladaptive T helper (Th)2 responses to inhaled allergens, which in turn lead to airway eosinophilia and airway hyperresponsiveness. However, many individuals with asthma have airway inflammation that is predominantly neutrophilic and resistant to treatment with inhaled glucocorticoids. An improved understanding of the molecular basis of this form of asthma might lead to improved strategies for its treatment. Here, we identify novel roles of the adaptor protein, TRIF, in neutrophilic responses to inhaled allergens. In different mouse models of asthma, Trif-deficient animals had marked reductions in interleukin (IL)-17, airway neutrophils, and airway hyperresponsiveness compared with WT mice, whereas airway eosinophils were generally similar in these two strains. Compared with lung dendritic cells (DCs) from WT mice, lung DCs from Trif-deficient mice displayed impaired LPS-induced migration to regional lymph nodes, lower levels of the costimulatory molecule, CD40, and produced smaller amounts of the Th17-promoting cytokines, IL-6 and IL-1β. When cultured with allergen-specific, naïve T cells, Trif-deficient lung DCs stimulated robust Th2 cell differentiation, but very weak Th1 and Th17 cell differentiation. Together, these findings reveal a TRIF-CD40-Th17 axis in the development of IL-17-associated, neutrophilic asthma.
doi:10.1038/mi.2014.56
PMCID: PMC4267961  PMID: 24985082
10.  Contribution of the Interaction of Streptococcus mutans Serotype k Strains with Fibrinogen to the Pathogenicity of Infective Endocarditis 
Infection and Immunity  2014;82(12):5223-5234.
Streptococcus mutans, a pathogen responsible for dental caries, is occasionally isolated from the blood of patients with bacteremia and infective endocarditis (IE). Our previous study demonstrated that serotype k-specific bacterial DNA is frequently detected in S. mutans-positive heart valve specimens extirpated from IE patients. However, the reason for this frequent detection remains unknown. In the present study, we analyzed the virulence of IE from S. mutans strains, focusing on the characterization of serotype k strains, most of which are positive for the 120-kDa cell surface collagen-binding protein Cbm and negative for the 190-kDa protein antigen (PA) known as SpaP, P1, antigen I/II, and other designations. Fibrinogen-binding assays were performed with 85 clinical strains classified by Cbm and PA expression levels. The Cbm+/PA− group strains had significantly higher fibrinogen-binding rates than the other groups. Analysis of platelet aggregation revealed that SA31, a Cbm+/PA− strain, induced an increased level of aggregation in the presence of fibrinogen, while negligible aggregation was induced by the Cbm-defective isogenic mutant SA31CBD. A rat IE model with an artificial impairment of the aortic valve created using a catheter showed that extirpated heart valves in the SA31 group displayed a prominent vegetation mass not seen in those in the SA31CBD group. These findings could explain why Cbm+/PA− strains are highly virulent and are related to the development of IE, and the findings could also explain the frequent detection of serotype k DNA in S. mutans-positive heart valve clinical specimens.
doi:10.1128/IAI.02164-14
PMCID: PMC4249299  PMID: 25287921
12.  Mitochondrial Ca2+ uptake by the voltage-dependent anion channel 2 regulates cardiac rhythmicity 
eLife  null;4:e04801.
Tightly regulated Ca2+ homeostasis is a prerequisite for proper cardiac function. To dissect the regulatory network of cardiac Ca2+ handling, we performed a chemical suppressor screen on zebrafish tremblor embryos, which suffer from Ca2+ extrusion defects. Efsevin was identified based on its potent activity to restore coordinated contractions in tremblor. We show that efsevin binds to VDAC2, potentiates mitochondrial Ca2+ uptake and accelerates the transfer of Ca2+ from intracellular stores into mitochondria. In cardiomyocytes, efsevin restricts the temporal and spatial boundaries of Ca2+ sparks and thereby inhibits Ca2+ overload-induced erratic Ca2+ waves and irregular contractions. We further show that overexpression of VDAC2 recapitulates the suppressive effect of efsevin on tremblor embryos whereas VDAC2 deficiency attenuates efsevin's rescue effect and that VDAC2 functions synergistically with MCU to suppress cardiac fibrillation in tremblor. Together, these findings demonstrate a critical modulatory role for VDAC2-dependent mitochondrial Ca2+ uptake in the regulation of cardiac rhythmicity.
DOI: http://dx.doi.org/10.7554/eLife.04801.001
eLife digest
The heart is a large muscle that pumps blood around the body by maintaining a regular rhythm of contraction and relaxation. If the heart loses this regular rhythm it works less efficiently, which can lead to life-threatening conditions.
Regular heart rhythms are maintained by changes in the concentration of calcium ions in the cytoplasm of the heart muscle cells. These changes are synchronised so that the heart cells contract in a controlled manner. In each cell, a contraction begins when calcium ions from outside the cell enter the cytoplasm by passing through a channel protein in the membrane that surrounds the cell. This triggers the release of even more calcium ions into the cytoplasm from stores within the cell. For the cells to relax, the calcium ions must then be pumped out of the cytoplasm to lower the calcium ion concentration back to the original level.
Shimizu et al. studied a zebrafish mutant—called tremblor—that has irregular heart rhythms because its heart muscle cells are unable to efficiently remove calcium ions from the cytoplasm. Embryos of the tremblor mutant were treated with a wide variety of chemical compounds with the aim of finding some that could correct the heart defect.
A compound called efsevin restores regular heart rhythms in tremblor mutants. Efsevin binds to a pump protein called VDAC2, which is found in compartments called mitochondria within the cell. Although mitochondria are best known for their role in supplying energy for the cell, they also act as internal stores for calcium. By binding to VDAC2, efsevin increases the rate at which calcium ions are pumped from the cytoplasm into the mitochondria. This restores rhythmic calcium ion cycling in the cytoplasm and enables the heart muscle cells to develop regular rhythms of contraction and relaxation. Increasing the levels of VDAC2 or another similar calcium ion pump protein in the heart cells can also restore a regular heart rhythm.
Efsevin can also correct irregular heart rhythms in human and mouse heart muscle cells, therefore the new role for mitochondria in controlling heart rhythms found by Shimizu et al. appears to be shared in other animals. The experiments have also identified the VDAC family of proteins as potential new targets for drug therapies to treat people with irregular heart rhythms.
DOI: http://dx.doi.org/10.7554/eLife.04801.002
doi:10.7554/eLife.04801
PMCID: PMC4293673  PMID: 25588501
mitochondria; arrhythmia; calcium handling; heart; VDAC; fibrillation; human; mouse; zebrafish
13.  EZH2 Protects Glioma Stem Cells from Radiation-Induced Cell Death in a MELK/FOXM1-Dependent Manner 
Stem Cell Reports  2015;4(2):226-238.
Summary
Glioblastoma (GBM)-derived tumorigenic stem-like cells (GSCs) may play a key role in therapy resistance. Previously, we reported that the mitotic kinase MELK binds and phosphorylates the oncogenic transcription factor FOXM1 in GSCs. Here, we demonstrate that the catalytic subunit of Polycomb repressive complex 2, EZH2, is targeted by the MELK-FOXM1 complex, which in turn promotes resistance to radiation in GSCs. Clinically, EZH2 and MELK are coexpressed in GBM and significantly induced in postirradiation recurrent tumors whose expression is inversely correlated with patient prognosis. Through a gain-and loss-of-function study, we show that MELK or FOXM1 contributes to GSC radioresistance by regulation of EZH2. We further demonstrate that the MELK-EZH2 axis is evolutionarily conserved in Caenorhabditis elegans. Collectively, these data suggest that the MELK-FOXM1-EZH2 signaling axis is essential for GSC radioresistance and therefore raise the possibility that MELK-FOXM1-driven EZH2 signaling can serve as a therapeutic target in irradiation-resistant GBM tumors.
Highlights
•EZH2 and MELK are coexpressed in GBM and post-IR recurrent tumors•MELK-mediated EZH2 is required for GSC radioresistance•MELK/EZH2 functions in radioresistance are evolutionarily conserved
In this article, Nakano and colleagues show that the catalytic subunit of Polycomb repressive complex 2, EZH2, is targeted by the MELK-FOXM1 complex, which in turn promotes resistance to radiation in GSCs. Additionally, they demonstrate that the MELK-EZH2 axis is evolutionarily conserved in Caenorhabditis elegans.
doi:10.1016/j.stemcr.2014.12.006
PMCID: PMC4325196  PMID: 25601206
14.  Identification and Functional Analysis of an Ammonium Transporter in Streptococcus mutans 
PLoS ONE  2014;9(9):e107569.
Streptococcus mutans, a Gram-positive bacterium, is considered to be a major etiologic agent of human dental caries and reported to form biofilms known as dental plaque on tooth surfaces. This organism is also known to possess a large number of transport proteins in the cell membrane for export and import of molecules. Nitrogen is an essential nutrient for Gram-positive bacteria, though alternative sources such as ammonium can also be utilized. In order to obtain nitrogen for macromolecular synthesis, nitrogen-containing compounds must be transported into the cell. However, the ammonium transporter in S. mutans remains to be characterized. The present study focused on characterizing the ammonium transporter gene of S. mutans and its operon, while related regulatory genes were also analyzed. The SMU.1658 gene corresponding to nrgA in S. mutans is homologous to the ammonium transporter gene in Bacillus subtilis and SMU.1657, located upstream of the nrgA gene and predicted to be glnB, is a member of the PII protein family. Using a nrgA-deficient mutant strain (NRGD), we examined bacterial growth in the presence of ammonium, calcium chloride, and manganese sulfate. Fluorescent efflux assays were also performed to reveal export molecules associated with the ammonium transporter. The growth rate of NRGD was lower, while its fluorescent intensity was much higher as compared to the parental strain. In addition, confocal laser scanning microscopy revealed that the structure of biofilms formed by NRGD was drastically different than that of the parental strain. Furthermore, transcriptional analysis showed that the nrgA gene was co-transcribed with the glnB gene. These results suggest that the nrgA gene in S. mutans is essential for export of molecules and biofilm formation.
doi:10.1371/journal.pone.0107569
PMCID: PMC4167856  PMID: 25229891
15.  Rigid microenvironments promote cardiac differentiation of mouse and human embryonic stem cells 
While adult heart muscle is the least regenerative of tissues, embryonic cardiomyocytes are proliferative, with embryonic stem (ES) cells providing an endless reservoir. In addition to secreted factors and cell-cell interactions, the extracellular microenvironment has been shown to play an important role in stem cell lineage specification, and understanding how scaffold elasticity influences cardiac differentiation is crucial to cardiac tissue engineering. Though previous studies have analyzed the role of the matrix elasticity on the function of differentiated cardiomyocytes, whether it affects the induction of cardiomyocytes from pluripotent stem cells is poorly understood. Here, we examined the role of matrix rigidity on the cardiac differentiation using mouse and human ES cells. Culture on polydimethylsiloxane (PDMS) substrates of varied monomer-to-crosslinker ratios revealed that rigid extracellular matrices promote a higher yield of de novo cardiomyocytes from undifferentiated ES cells. Using an genetically modified ES system that allows us to purify differentiated cardiomyocytes by drug selection, we demonstrate that rigid environments induce higher cardiac troponin T expression, beating rate of foci, and expression ratio of adult α- to fetal β- myosin heavy chain in a purified cardiac population. M-mode and mechanical interferometry image analyses demonstrate that these ES-derived cardiomyocytes display functional maturity and synchronization of beating when co-cultured with neonatal cardiomyocytes harvested from a developing embryo. Together, these data identify matrix stiffness as an independent factor that instructs not only the maturation of the already differentiated cardiomyocytes but also the induction and proliferation of cardiomyocytes from undifferentiated progenitors. Manipulation of the stiffness will help direct the production of functional cardiomyocytes en masse from stem cells for regenerative medicine purposes.
doi:10.1088/1468-6996/14/2/025003
PMCID: PMC3845966  PMID: 24311969
Cardiac Differentiation; Pluripotent Embryonic Stem Cell; Matrix elasticity; Drug-selected cardiomyocyte; Synchronization; Mechanical interferometry
16.  Ethics of iPSC-Based Clinical Research for Age-Related Macular Degeneration: Patient-Centered Risk-Benefit Analysis 
Stem Cell Reviews  2014;10(6):743-752.
The opportunity to undergo an induced pluripotent stem cell-based autologous transplant can strike patients as a chance for a cure from a debilitating condition with few options for respite. However, when clinical studies of this caliber present themselves, patients and researchers, each with their own set of motives, may find it difficult to take a balanced approach to evaluating them. We present a patient-centered risk-benefit analysis of the iPSC-based clinical research currently underway in Japan, including a survey of in vitro and in vivo tests that support this project, an in-depth discussion of risks, and further elucidation of considerations patients may wish to consider. The arguments presented will assist patients in undertaking a more informed decision-making process.
doi:10.1007/s12015-014-9536-x
PMCID: PMC4225048  PMID: 24974102
Human subject research; Induced pluripotent stem cells (iPSCs); Age-related macular degeneration (AMD); Patient-centered risk-benefit analysis; Tumorigenicity; Therapeutic misconception/misestimation
17.  Multi-Kinase Inhibitor C1 Triggers Mitotic Catastrophe of Glioma Stem Cells Mainly through MELK Kinase Inhibition 
PLoS ONE  2014;9(4):e92546.
Glioblastoma multiforme (GBM) is a highly lethal brain tumor. Due to resistance to current therapies, patient prognosis remains poor and development of novel and effective GBM therapy is crucial. Glioma stem cells (GSCs) have gained attention as a therapeutic target in GBM due to their relative resistance to current therapies and potent tumor-initiating ability. Previously, we identified that the mitotic kinase maternal embryonic leucine-zipper kinase (MELK) is highly expressed in GBM tissues, specifically in GSCs, and its expression is inversely correlated with the post-surgical survival period of GBM patients. In addition, patient-derived GSCs depend on MELK for their survival and growth both in vitro and in vivo. Here, we demonstrate evidence that the role of MELK in the GSC survival is specifically dependent on its kinase activity. With in silico structure-based analysis for protein-compound interaction, we identified the small molecule Compound 1 (C1) is predicted to bind to the kinase-active site of MELK protein. Elimination of MELK kinase activity was confirmed by in vitro kinase assay in nano-molar concentrations. When patient-derived GSCs were treated with C1, they underwent mitotic arrest and subsequent cellular apoptosis in vitro, a phenotype identical to that observed with shRNA-mediated MELK knockdown. In addition, C1 treatment strongly induced tumor cell apoptosis in slice cultures of GBM surgical specimens and attenuated growth of mouse intracranial tumors derived from GSCs in a dose-dependent manner. Lastly, C1 treatment sensitizes GSCs to radiation treatment. Collectively, these data indicate that targeting MELK kinase activity is a promising approach to attenuate GBM growth by eliminating GSCs in tumors.
doi:10.1371/journal.pone.0092546
PMCID: PMC3989164  PMID: 24739874
18.  Complete Atrial-Specific Knockout of Sodium-Calcium Exchange Eliminates Sinoatrial Node Pacemaker Activity 
PLoS ONE  2013;8(11):e81633.
The origin of sinoatrial node (SAN) pacemaker activity in the heart is controversial. The leading candidates are diastolic depolarization by “funny” current (If) through HCN4 channels (the “Membrane Clock“ hypothesis), depolarization by cardiac Na-Ca exchange (NCX1) in response to intracellular Ca cycling (the "Calcium Clock" hypothesis), and a combination of the two (“Coupled Clock”). To address this controversy, we used Cre/loxP technology to generate atrial-specific NCX1 KO mice. NCX1 protein was undetectable in KO atrial tissue, including the SAN. Surface ECG and intracardiac electrograms showed no atrial depolarization and a slow junctional escape rhythm in KO that responded appropriately to β-adrenergic and muscarinic stimulation. Although KO atria were quiescent they could be stimulated by external pacing suggesting that electrical coupling between cells remained intact. Despite normal electrophysiological properties of If in isolated patch clamped KO SAN cells, pacemaker activity was absent. Recurring Ca sparks were present in all KO SAN cells, suggesting that Ca cycling persists but is uncoupled from the sarcolemma. We conclude that NCX1 is required for normal pacemaker activity in murine SAN.
doi:10.1371/journal.pone.0081633
PMCID: PMC3836769  PMID: 24278453
19.  Scl represses cardiomyogenesis in prospective hemogenic endothelium and endocardium 
Cell  2012;150(3):590-605.
Summary
Endothelium in embryonic hematopoietic tissues generates hematopoietic stem/progenitor cells; however, it is unknown how its unique potential is specified. We show that transcription factor Scl/Tal1 is essential for both establishing the hematopoietic transcriptional program in hemogenic endothelium and preventing its misspecification to a cardiomyogenic fate. Scl−/− embryos activated a cardiac transcriptional program in yolk sac endothelium, leading to the emergence of CD31+Pdgfrα+ cardiogenic precursors that generated spontaneously beating cardiomyocytes. Ectopic cardiogenesis was also observed in Scl−/− hearts, where the disorganized endocardium precociously differentiated into cardiomyocytes. Induction of mosaic deletion of Scl in Sclfl/fl Rosa26Cre-ERT2 embryos revealed a cell-intrinsic, temporal requirement for Scl to prevent cardiomyogenesis from endothelium. Scl−/− endothelium also upregulated the expression of Wnt antagonists, which promoted rapid cardiomyocyte differentiation of ectopic cardiogenic cells. These results reveal unexpected plasticity in embryonic endothelium such that loss of a single master regulator can induce ectopic cardiomyogenesis from endothelial cells.
doi:10.1016/j.cell.2012.06.026
PMCID: PMC3624753  PMID: 22863011
20.  Migratory properties of pulmonary dendritic cells are determined by their developmental lineage 
Mucosal immunology  2012;6(4):678-691.
The chemokine receptor, CCR7, directs the migration of dendritic cells (DCs) from peripheral tissue to draining lymph nodes (LNs). However, it is unknown whether all pulmonary DCs possess migratory potential. Using novel Ccr7gfp reporter mice, we found that Ccr7 is expressed in CD103+ and a CD14med/lo subset of CD11bhi classical (c) DCs but not in monocyte-derived (mo) DCs, including Ly-6ChiCD11bhi inflammatory DCs and CD14hiCD11bhi DCs. Consequently, cDCs migrated to lung-draining LNs but moDCs did not. Mice lacking the chemokine receptor, CCR2, also lacked inflammatory DCs in the lung after lipopolysaccharide inhalation but retained normal levels of migratory DCs. Conversely, the lungs of fms-like tyrosine kinase 3 ligand (Flt3L)-deficient mice lacked cDCs but retained moDCs, which were functionally mature but did not express Ccr7 and were uniformly non-migratory. Thus, the migratory properties of pulmonary DCs are determined by their developmental lineage.
doi:10.1038/mi.2012.106
PMCID: PMC3652913  PMID: 23168837
21.  Pulmonary CD103+ dendritic cells prime Th2 responses to inhaled allergens 
Mucosal immunology  2011;5(1):53-65.
Allergic asthma stems largely from the actions of T helper 2 (Th2) cells, but the pathways that initiate Th2 responses to inhaled allergens are not fully understood. In the lung, there are two major subsets of dendritic cells (DCs), displaying CD11b or CD103. We found that after taking up inhaled ovalbumin in vivo, purified CD103+ DCs from the lung or lung-draining lymph nodes primed Th2 differentiation ex vivo. Th2 induction by CD103+ DCs was also seen when cockroach or house dust mite allergens were used. In contrast, CD11bhi DCs primed Th1 differentiation. Moreover, mice lacking CD103+ DCs displayed diminished Th2 priming to various inhaled allergens and did not develop asthma-like responses following subsequent allergen challenge. Low-level antigen presentation by CD103+ DCs was necessary, but not sufficient for Th2 priming. Together, these findings show that CD103+ DCs have a significant role in priming Th2 responses to inhaled allergens.
doi:10.1038/mi.2011.47
PMCID: PMC3697034  PMID: 22012243
22.  Polychlorinated biphenyl (118) activates osteoclasts and induces bone resorption in goldfish 
To analyze the effect of polychlorinated biphenyl (PCB) 118 on fish bone metabolism, we examined osteoclastic and osteoblastic activities, as well as plasma calcium levels, in the scales of PCB (118)-injected goldfish. In addition, effect of PCB (118) on osteoclasts and osteoblasts was investigated in vitro. Immature goldfish, in which the endogenous effects of sex steroids are negligible, were used. PCB (118) was solubilized in dimethyl sulfoxide at a concentration of 10 ppm. At 1 and 2 days after PCB (118) injection (100 ng/g body weight), both osteoclastic and osteoblastic activities, and plasma calcium levels were measured. In an in vitro study, then, both osteoclastic and osteoblastic activities as well as each marker mRNA expression were examined. At 2 days, scale osteoclastic activity in PCB (118)-injected goldfish increased significantly, while osteoblastic activity did not change significantly. Corresponding to osteoclastic activity, plasma calcium levels increased significantly at 2 days after PCB (118) administration. Osteoclastic activation also occurred in the marker enzyme activities and mRNA expressions in vitro. Thus, we conclude that PCB (118) disrupts bone metabolism in goldfish both in vivo and in vitro experiments.
doi:10.1007/s11356-012-1347-5
PMCID: PMC4021165  PMID: 23247518
PCB (118); Bone metabolism; Fish scales; Osteoclasts; Osteoblasts; Plasma calcium
23.  Cardiac origin of smooth muscle cells in the inflow tract 
Multipotent Isl1+ heart progenitors give rise to three major cardiovascular cell types; cardiac, smooth muscle, and endothelial cells, and play a pivotal role in lineage diversification during cardiogenesis. A critical question is pinpointing when this cardiac-vascular lineage decision is made, and how this plasticity serves to coordinate cardiac chamber and vessel growth. The posterior domain of the Isl1-positive second heart field contributes to the SLN-positive atrial myocardium and myocardial sleeves in the cardiac inflow tract, where myocardial and vascular smooth muscle layers form anatomical and functional continuity. Herein, using a new atrial specific SLN-Cre knockin mouse line, we report that an Isl1+/SLN+ transient cell population contributes to cardiac as well as smooth muscle cells at the heart-vessel junction in cardiac inflow tract. The Isl1+/SLN+ cells are capable of giving rise to cardiac and smooth muscle cells until late gestational stages. These data suggest that the cardiac and smooth muscle cells in the cardiac inflow tract share a common developmental origin.
doi:10.1016/j.yjmcc.2010.10.009
PMCID: PMC3031779  PMID: 20974149
cardiogenesis; myogenic progenitor; smooth muscle; great vessel; plasticity
24.  IL-35 Production by ICOS+ Tregs Reverses Established IL-17-dependent Allergic Airways Disease 
Background
Recent evidence suggests that IL-17 contributes to airway hyperresponsiveness (AHR); however, the mechanisms that suppress the production of this cytokine remain poorly defined.
Objectives
We sought to understand the cellular and molecular basis for suppression of established, IL-17-dependent allergic airways disease.
Methods
Mice were sensitized by airway instillations of ovalbumin (OVA) together with low levels of lipopolysaccharide. Leukocyte recruitment to the lung and AHR were assessed following daily challenges with aerosolized OVA. Flow cytometry and gene targeted mice were used to identify naturally-arising subsets of regulatory T cells (Tregs) and their cytokines required for the suppression of established allergic airway disease.
Results
Allergic sensitization through the airway primed both effector and regulatory responses. Effector responses were initially dominant and led to airway inflammation and IL-17-dependent AHR. However, after multiple daily allergen challenges, IL-17 production and AHR declined, even though pulmonary levels of Th17 cells remained high. This loss of AHR was reversible and required the expansion of a Treg subset expressing both Foxp3 and inducible co-stimulator (ICOS). These Tregs also expressed the regulatory cytokines, IL-10, TGF-beta and IL-35. Whereas IL-10 and TGF-beta were dispensable for suppression of airway hyperresponsiveness, IL-35 was required. Analysis of human ICOS+ Tregs revealed that they also selectively expressed IL-35.
Conclusion
IL-35 production by ICOS+ Tregs can suppress IL-17 production and thereby reverse established, IL-17-dependent AHR in mice. The production of IL-35 by human ICOS+ Tregs suggests that targeting this pathway might be of therapeutic value for treating allergic asthma in humans.
doi:10.1016/j.jaci.2011.08.009
PMCID: PMC3269135  PMID: 21906794
Asthma; airway hyperresponsiveness; AHR; IL-17; Th17; Th2; IL-35; ICOS; ovalbumin
25.  Atypical gaze patterns in children and adults with autism spectrum disorders dissociated from developmental changes in gaze behaviour 
Eye tracking has been used to investigate gaze behaviours in individuals with autism spectrum disorder (ASD). However, traditional analysis has yet to find behavioural characteristics shared by both children and adults with ASD. To distinguish core ASD gaze behaviours from those that change with development, we examined temporo-spatial gaze patterns in children and adults with and without ASD while they viewed video clips. We summarized the gaze patterns of 104 participants using multidimensional scaling so that participants with similar gaze patterns would cluster together in a two-dimensional plane. Control participants clustered in the centre, reflecting a standard gaze behaviour, whereas participants with ASD were distributed around the periphery. Moreover, children and adults were separated on the plane, thereby showing a clear effect of development on gaze behaviours. Post hoc frame-by-frame analyses revealed the following findings: (i) both ASD groups shifted their gaze away from a speaker earlier than the control groups; (ii) both ASD groups showed a particular preference for letters; and (iii) typical infants preferred to watch the mouth rather than the eyes during speech, a preference that reversed with development. These results highlight the importance of taking the effect of development into account when addressing gaze behaviours characteristic of ASD.
doi:10.1098/rspb.2010.0587
PMCID: PMC2982027  PMID: 20484237
eye tracking; eye movements; autism; development; mouth viewing; turn taking

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