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Find video protocols related to scientific articles indexed in Pubmed.
TNF-mediated inflammation represses GATA1 and activates p38 MAP kinase in RPS19 deficient hematopoietic progenitors.
Blood
PUBLISHED: 10-02-2014
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Diamond-Blackfan Anemia (DBA) is an inherited disorder, characterized by defects in erythropoiesis, congenital abnormalities, and predisposition to cancer. Approximately 25% of DBA patients have a mutation in RPS19, which encodes a component of the 40S ribosomal subunit. Upregulation of p53 contributes to the pathogenesis of DBA, but the link between ribosomal protein mutations and erythropoietic defects is not well understood. We found that RPS19 deficiency in hematopoietic progenitor cells leads to decreased GATA1 expression in the erythroid progenitor population and p53-dependent upregulation of TNF-? in non-erythroid cells. The decrease in GATA1 expression was mediated, at least in part, by activation of p38 MAPK in erythroid cells and rescued by inhibition of TNF-? or p53. The anemia phenotype in RPS19 deficient zebrafish was reversed by treatment with the TNF-? inhibitor etanercept. Our data reveal that RPS19 deficiency leads to inflammation, p53-dependent increase in TNF-?, activation of p38 MAPK, and decreased GATA1 expression, suggesting a novel mechanism for the erythroid defects observed in DBA.
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Vanishing Point-Based Image Transforms for Enhancement of Probabilistic Occupancy Map-Based People Localization.
IEEE Trans Image Process
PUBLISHED: 09-25-2014
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The widespread use of vision-based surveillance systems has inspired many research efforts on people localization. In this paper, a series of novel image transforms based on the vanishing point of vertical lines are proposed for enhancement of the probabilistic occupancy map (POM) based people localization scheme presented in [1]. Utilizing the characteristic that the extensions of vertical lines intersect at a vanishing point, the proposed transforms, based on image or ground plane coordinate system, aims at producing transformed images wherein each standing/walking person will have an upright appearance. Thus, the degradation in localization accuracy due to the deviation of camera configuration constraint specified in [1] can be alleviated, while the computation efficiency resulted from the applicability of integral image can be retained. Experimental results show that significant improvement in POM-based people localization for more general camera configurations can indeed be achieved with the proposed image transforms.
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The Translation Initiation Factor eIF3i Up-regulates Vascular Endothelial Growth Factor A, Accelerates Cell Proliferation, and Promotes Angiogenesis in Embryonic Development and Tumorigenesis.
J. Biol. Chem.
PUBLISHED: 08-21-2014
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Vascular endothelial growth factor A (VEGFA) is a critical proangiogenic factor that is activated by hypoxia at both the transcriptional and post-transcriptional levels. In hypoxia conditions, stabilized hypoxia-inducible factor 1? (HIF1A) is the key regulator for transcriptional activation of VEGFA. However, the post-transcriptional control of VEGFA expression remains poorly understood. Here, we report that the eukaryotic translation initiation factor 3i (eIF3i) is required for VEGFA protein expression in both normal embryonic and tumorigenic angiogenesis. eIF3i is dynamically expressed in the early stages of zebrafish embryogenesis and in human hepatocellular carcinoma tissues. eIF3i homozygous mutant zebrafish embryos show severe angiogenesis defects and human hepatocellular cancer cells with depletion of eIF3i to induce less angiogenesis in tumor models. Under hypoxia, the HIF1A protein can interact with its binding sequence in the eIF3i promoter and activate eIF3i transcription. The expression of VEGFA, which should rise in hypoxia, is significantly inhibited by eIF3i siRNA treatment. Moreover, eIF3i knockdown did not cause a general translation repression but specifically reduced the translation efficiency of the VEGFA mRNAs. Taken together, our results suggest that eIF3i is induced by HIF1A under hypoxia and controls normal and tumorigenic angiogenesis through regulating VEGFA protein translation.
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The heparan sulfate proteoglycan agrin contributes to barrier properties of mouse brain endothelial cells by stabilizing adherens junctions.
Cell Tissue Res.
PUBLISHED: 08-09-2014
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Barrier characteristics of brain endothelial cells forming the blood-brain barrier (BBB) are tightly regulated by cellular and acellular components of the neurovascular unit. During embryogenesis, the accumulation of the heparan sulfate proteoglycan agrin in the basement membranes ensheathing brain vessels correlates with BBB maturation. In contrast, loss of agrin deposition in the vasculature of brain tumors is accompanied by the loss of endothelial junctional proteins. We therefore wondered whether agrin had a direct effect on the barrier characteristics of brain endothelial cells. Agrin increased junctional localization of vascular endothelial (VE)-cadherin, ?-catenin, and zonula occludens-1 (ZO-1) but not of claudin-5 and occludin in the brain endothelioma cell line bEnd5 without affecting the expression levels of these proteins. This was accompanied by an agrin-induced reduction of the paracellular permeability of bEnd5 monolayers. In vivo, the lack of agrin also led to reduced junctional localization of VE-cadherin in brain microvascular endothelial cells. Taken together, our data support the notion that agrin contributes to barrier characteristics of brain endothelium by stabilizing the adherens junction proteins VE-cadherin and ?-catenin and the junctional protein ZO-1 to brain endothelial junctions.
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Tcf7l2 is required for left-right asymmetric differentiation of habenular neurons.
Curr. Biol.
PUBLISHED: 06-11-2014
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Although left-right asymmetries are common features of nervous systems, their developmental bases are largely unknown. In the zebrafish epithalamus, dorsal habenular neurons adopt medial (dHbm) and lateral (dHbl) subnuclear character at very different frequencies on the left and right sides. The left-sided parapineal promotes the elaboration of dHbl character in the left habenula, albeit by an unknown mechanism. Likewise, the genetic pathways acting within habenular neurons to control their asymmetric differentiated character are unknown.
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Effects of L-carnitine supplementation on oxidative stress and antioxidant enzymes activities in patients with coronary artery disease: a randomized, placebo-controlled trial.
Nutr J
PUBLISHED: 05-19-2014
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Cardiovascular disease is the leading cause of death worldwide. Higher oxidative stress may contribute to the pathogenesis of coronary artery disease (CAD). The purpose of this study was to investigate the effect of L-carnitine (LC, 1000 mg/d) on the markers of oxidative stress and antioxidant enzymes activities in CAD patients.
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The role of the DNA damage response in zebrafish and cellular models of Diamond Blackfan anemia.
Dis Model Mech
PUBLISHED: 05-08-2014
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Ribosomal biogenesis involves the processing of pre-ribosomal RNA. A deficiency of some ribosomal proteins (RPs) impairs processing and causes Diamond Blackfan anemia (DBA), which is associated with anemia, congenital malformations and cancer. p53 mediates many features of DBA, but the mechanism of p53 activation remains unclear. Another hallmark of DBA is the upregulation of adenosine deaminase (ADA), indicating changes in nucleotide metabolism. In RP-deficient zebrafish, we found activation of both nucleotide catabolism and biosynthesis, which is consistent with the need to break and replace the faulty ribosomal RNA. We also found upregulation of deoxynucleotide triphosphate (dNTP) synthesis - a typical response to replication stress and DNA damage. Both RP-deficient zebrafish and human hematopoietic cells showed activation of the ATR/ATM-CHK1/CHK2/p53 pathway. Other features of RP deficiency included an imbalanced dNTP pool, ATP depletion and AMPK activation. Replication stress and DNA damage in cultured cells in non-DBA models can be decreased by exogenous nucleosides. Therefore, we treated RP-deficient zebrafish embryos with exogenous nucleosides and observed decreased activation of p53 and AMPK, reduced apoptosis, and rescue of hematopoiesis. Our data suggest that the DNA damage response contributes to p53 activation in cellular and zebrafish models of DBA. Furthermore, the rescue of RP-deficient zebrafish with exogenous nucleosides suggests that nucleoside supplements could be beneficial in the treatment of DBA.
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A simple strategy for in situ fabrication of a smart hydrogel microvalve within microchannels for thermostatic control.
Lab Chip
PUBLISHED: 05-08-2014
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Self-regulation of temperature in microchip systems is crucial for their applications in biomedical fields such as cell culture and biomolecule synthesis as well as those cases that require constant temperature conditions. Here we report on a simple and versatile approach for in situ fabrication of a smart hydrogel microvalve within a microchip for thermostatic control. The thermo-responsive hydrogel microvalve enables the "on-off" switch by sensing temperature fluctuations to control the fluid flux as well as the fluid heat exchange for self-regulation of the temperature at a constant range. Such temperature self-regulation is demonstrated by integrating the microvalve-incorporated microchip into the flow circulation loop of a micro-heat-exchanging system for thermostatic control. Moreover, the microvalve-incorporated microchip is employed for culturing cells under temperature self-regulation. The smart microvalve shows great potential as a temperature controller for applications that require thermostatic conditions. This approach offers a facile and flexible strategy for in situ fabricating hydrogel microvalves within microchips as chemostats and microreactors for biomedical applications.
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Aspect ratio plays a role in the hazard potential of CeO2 nanoparticles in mouse lung and zebrafish gastrointestinal tract.
ACS Nano
PUBLISHED: 04-16-2014
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We have previously demonstrated that there is a relationship between the aspect ratio (AR) of CeO2 nanoparticles and in vitro hazard potential. CeO2 nanorods with AR ? 22 induced lysosomal damage and progressive effects on IL-1? production and cytotoxicity in the human myeloid cell line, THP-1. In order to determine whether this toxicological paradigm for long aspect ratio (LAR) CeO2 is also relevant in vivo, we performed comparative studies in the mouse lung and gastrointestinal tract (GIT) of zebrafish larvae. Although oropharyngeal aspiration could induce acute lung inflammation for CeO2 nanospheres and nanorods, only the nanorods with the highest AR (C5) induced significant IL-1? and TGF-?1 production in the bronchoalveolar lavage fluid at 21 days but did not induce pulmonary fibrosis. However, after a longer duration (44 days) exposure to 4 mg/kg of the C5 nanorods, more collagen production was seen with CeO2 nanorods vs nanospheres after correcting for Ce lung burden. Using an oral-exposure model in zebrafish larvae, we demonstrated that C5 nanorods also induced significant growth inhibition, a decrease in body weight, and delayed vertebral calcification. In contrast, CeO2 nanospheres and shorter nanorods had no effect. Histological and transmission electron microscopy analyses showed that the key injury mechanism of C5 was in the epithelial lining of the GIT, which demonstrated blunted microvilli and compromised digestive function. All considered, these data demonstrate that, similar to cellular studies, LAR CeO2 nanorods exhibit more toxicity in the lung and GIT, which could be relevant to inhalation and environmental hazard potential.
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Heme acts through the Bach1b/Nrf2a-MafK pathway to regulate exocrine peptidase precursor genes in porphyric zebrafish.
Dis Model Mech
PUBLISHED: 03-20-2014
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Using a zebrafish model of hepatoerythropoietic porphyria (HEP), we identify a previously unknown mechanism underlying heme-mediated regulation of exocrine zymogens. Zebrafish bach1b, nrf2a and mafK are all expressed in the zebrafish exocrine pancreas. Overexpression of bach1b or knockdown of nrf2a result in the downregulation of the expression of the exocrine zymogens, whereas overexpression of nrf2a or knockdown of bach1b cause their upregulation. In vitro luciferase assays demonstrate that heme activates the zymogens in a dosage-dependent manner and that the zymogen promoter activities require the integral Maf recognition element (MARE) motif. The Bach1b-MafK heterodimer represses the zymogen promoters, whereas the Nrf2a-MafK heterodimer activates them. Furthermore, chromatin immunoprecipitation (ChIP) assays show that MafK binds to the MARE sites in the 5' regulatory regions of the zymogens. Taken together, these data indicate that heme stimulates the exchange of Bach1b for Nrf2a at MafK-occupied MARE sites and that, particularly in heme-deficient porphyria, the repressive Bach1b-MafK heterodimer dominates, which can be exchanged for the activating Nrf2a-MafK heterodimer upon treatment with hemin. These results provide novel insights into the regulation of exocrine function, as well as the pathogenesis of porphyria, and should be useful for designing new therapies for both types of disease.
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Differential regulation of AChR clustering in the polar and equatorial region of murine muscle spindles.
Eur. J. Neurosci.
PUBLISHED: 03-13-2014
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Intrafusal fibers of muscle spindles are innervated in the central region by afferent sensory axons and at both polar regions by efferent ?-motoneurons. We previously demonstrated that both neuron-muscle contact sites contain cholinergic synapse-like specialisation, including aggregates of the nicotinic acetylcholine receptor (AChR). In this study we tested the hypothesis that agrin and its receptor complex (consisting of LRP4 and the tyrosine kinase MuSK) are involved in the aggregation of AChRs in muscle spindles, similar to their role at the neuromuscular junction. We show that agrin, MuSK and LRP4 are concentrated at the contact site between the intrafusal fibers and the sensory- and ?-motoneuron, respectively, and that they are expressed in the cell bodies of proprioceptive neurons in dorsal root ganglia. Moreover, agrin and LRP4, but not MuSK, are expressed in ?-motoneuron cell bodies in the ventral horn of the spinal cord. In agrin- and in MuSK-deficient mice, AChR aggregates are absent from the polar regions. In contrast, the subcellular concentration of AChRs in the central region where the sensory neuron contacts the intrafusal muscle fiber is apparently unaffected. Skeletal muscle-specific expression of miniagrin in agrin(-/-) mice in vivo is sufficient to restore the formation of ?-motoneuron endplates. These results show that agrin and MuSK are major determinants during the formation of ?-motoneuron endplates but appear dispensable for the aggregation of AChRs at the central region. Our results therefore suggest different molecular mechanisms for AChR clustering within two domains of intrafusal fibers.
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GLUT3 gene expression is critical for embryonic growth, brain development and survival.
Mol. Genet. Metab.
PUBLISHED: 01-25-2014
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Glucose is the primary energy source for eukaryotic cells and the predominant substrate for the brain. GLUT3 is essential for trans-placental glucose transport and highly expressed in the mammalian brain. To further elucidate the role of GLUT3 in embryonic development, we utilized the vertebrate whole animal model system of Danio rerio as a tractable system for defining the cellular and molecular mechanisms altered by impaired glucose transport and metabolism related to perturbed expression of GLUT3. The comparable orthologue of human GLUT3 was identified and the expression of this gene abrogated during early embryonic development. In a dose-dependent manner embryonic brain development was disrupted resulting in a phenotype of aberrant brain organogenesis, associated with embryonic growth restriction and increased cellular apoptosis. Rescue of the morphant phenotype was achieved by providing exogenous GLUT3 mRNA. We conclude that GLUT3 is critically important for brain organogenesis and embryonic growth. Disruption of GLUT3 is responsible for the phenotypic spectrum of embryonic growth restriction to demise and neural apoptosis with microcephaly.
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Kctd10 regulates heart morphogenesis by repressing the transcriptional activity of Tbx5a in zebrafish.
Nat Commun
PUBLISHED: 01-17-2014
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The T-box transcription factor Tbx5 (Tbx5a in zebrafish) plays a crucial role in the formation of cardiac chambers in a dose-dependent manner. Its deregulation leads to congenital heart disease. However, little is known regarding its regulation. Here we isolate a zebrafish mutant with heart malformations, called 34c. The affected gene is identified as kctd10, a member of the potassium channel tetramerization domain (KCTD)-containing family. In the mutant, the expressions of the atrioventricular canal marker genes, such as tbx2b, hyaluronan synthase 2 (has2), notch1b and bmp4, are changed. The knockdown of tbx5 rescues the ectopic expression of has2, and knockdown of either tbx5a or has2 alleviates the heart defects. We show that Kctd10 directly binds to Tbx5 to repress its transcriptional activity. Our results reveal a new essential factor for cardiac development and suggest that KCTD10 could be considered as a new causative gene of congenital heart disease.
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CasOT: a genome-wide Cas9/gRNA off-target searching tool.
Bioinformatics
PUBLISHED: 01-02-2014
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The CRISPR/Cas or Cas9/guide RNA system is a newly developed, easily engineered and highly effective tool for gene targeting; it has considerable off-target effects in cultured human cells and in several organisms. However, the Cas9/guide RNA target site is too short for existing alignment tools to exhaustively and effectively identify potential off-target sites. CasOT is a local tool designed to find potential off-target sites in any given genome or user-provided sequence, with user-specified types of protospacer adjacent motif, and number of mismatches allowed in the seed and non-seed regions.
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The relationship between frontal lobe lesions, course of post-stroke depression, and 1-year prognosis in patients with first-ever ischemic stroke.
PLoS ONE
PUBLISHED: 01-01-2014
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Most studies on post-stroke depression (PSD) have focused on a certain time point after stroke instead of the time course of PSD. The aim of this study was to determine the relationship between frontal lobe lesions, course of PSD over a year following the stroke onset, and the 1-year prognosis in patients with first-ever ischemic stroke.
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Nom1 mediates pancreas development by regulating ribosome biogenesis in zebrafish.
PLoS ONE
PUBLISHED: 01-01-2014
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Ribosome biogenesis is an important biological process for proper cellular function and development. Defects leading to improper ribosome biogenesis can cause diseases such as Diamond-Blackfan anemia and Shwachman-Bodian-Diamond syndrome. Nucleolar proteins are a large family of proteins and are involved in many cellular processes, including the regulation of ribosome biogenesis. Through a forward genetic screen and positional cloning, we identified and characterized a zebrafish line carrying mutation in nucleolar protein with MIF4G domain 1 (nom1), which encodes a conserved nulceolar protein with a role in pre-rRNA processing. Zebrafish nom1 mutants exhibit major defects in endoderm development, especially in exocrine pancreas. Further studies revealed that impaired proliferation of ptf1a-expressing pancreatic progenitor cells mainly contributed to the phenotype. RNA-seq and molecular analysis showed that ribosome biogenesis and pre-mRNA splicing were both affected in the mutant embryos. Several defects of ribosome assembly have been shown to have a p53-dependent mechanism. In the nom1 mutant, loss of p53 did not rescue the pancreatic defect, suggesting a p53-independent role. Further studies indicate that protein phosphatase 1 alpha, an interacting protein to Nom1, could partially rescue the pancreatic defect in nom1 morphants if a human nucleolar localization signal sequence was artificially added. This suggests that targeting Pp1? into the nucleolus by Nom1 is important for pancreatic proliferation. Altogether, our studies revealed a new mechanism involving Nom1 in controlling vertebrate exocrine pancreas formation.
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Injection of a soluble fragment of neural agrin (NT-1654) considerably improves the muscle pathology caused by the disassembly of the neuromuscular junction.
PLoS ONE
PUBLISHED: 01-01-2014
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Treatment of neuromuscular diseases is still an unsolved problem. Evidence over the last years strongly indicates the involvement of malformation and dysfunction of neuromuscular junctions in the development of such medical conditions. Stabilization of NMJs thus seems to be a promising approach to attenuate the disease progression of muscle wasting diseases. An important pathway for the formation and maintenance of NMJs is the agrin/Lrp4/MuSK pathway. Here we demonstrate that the agrin biologic NT-1654 is capable of activating the agrin/Lrp4/MuSK system in vivo, leading to an almost full reversal of the sarcopenia-like phenotype in neurotrypsin-overexpressing (SARCO) mice. We also show that injection of NT-1654 accelerates muscle re-innervation after nerve crush. This report demonstrates that a systemically administered agrin fragment has the potential to counteract the symptoms of neuromuscular disorders.
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Syntheses of Fused Tetracyclic Quinolines via Ugi-Variant MCR and Pd-Catalyzed Bis-annulation.
J. Org. Chem.
PUBLISHED: 12-13-2013
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Diversity-oriented synthesis of fused tetracyclic 6,11-dihydroquinoxalino[2,3-b]quinolines is described via a sequential Ugi-variant multicomponent reaction and Pd-catalyzed bis-annulation in one-pot process.
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[Molecular epidemiological analysis of ?- and ?-thalassemia in Fujian province].
Zhonghua Yi Xue Yi Chuan Xue Za Zhi
PUBLISHED: 08-09-2013
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To investigate the gene prevalence and spectrum of alpha- and beta-thalassemia in Fujian province.
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Zebrafish tyrosine hydroxylase 2 gene encodes tryptophan hydroxylase.
J. Biol. Chem.
PUBLISHED: 06-10-2013
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The primary pathological hallmark of Parkinson disease (PD) is the profound loss of dopaminergic neurons in the substantia nigra pars compacta. To facilitate the understanding of the underling mechanism of PD, several zebrafish PD models have been generated to recapitulate the characteristics of dopaminergic (DA) neuron loss. In zebrafish studies, tyrosine hydroxylase 1 (th1) has been frequently used as a molecular marker of DA neurons. However, th1 also labels norepinephrine and epinephrine neurons. Recently, a homologue of th1, named tyrosine hydroxylase 2 (th2), was identified based on the sequence homology and subsequently used as a novel marker of DA neurons. In this study, we present evidence that th2 co-localizes with serotonin in the ventral diencephalon and caudal hypothalamus in zebrafish embryos. In addition, knockdown of th2 reduces the level of serotonin in the corresponding th2-positive neurons. This phenotype can be rescued by both zebrafish th2 and mouse tryptophan hydroxylase 1 (Tph1) mRNA as well as by 5-hydroxytryptophan, the product of tryptophan hydroxylase. Moreover, the purified Th2 protein has tryptophan hydroxylase activity comparable with that of the mouse TPH1 protein in vitro. Based on these in vivo and in vitro results, we conclude that th2 is a gene encoding for tryptophan hydroxylase and should be used as a marker gene of serotonergic neurons.
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Chromosomal deletions and inversions mediated by TALENs and CRISPR/Cas in zebrafish.
Nucleic Acids Res.
PUBLISHED: 06-06-2013
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Customized TALENs and Cas9/gRNAs have been used for targeted mutagenesis in zebrafish to induce indels into protein-coding genes. However, indels are usually not sufficient to disrupt the function of non-coding genes, gene clusters or regulatory sequences, whereas large genomic deletions or inversions are more desirable for this purpose. By injecting two pairs of TALEN mRNAs or two gRNAs together with Cas9 mRNA targeting distal DNA sites of the same chromosome, we obtained predictable genomic deletions or inversions with sizes ranging from several hundred bases to nearly 1 Mb. We have successfully achieved this type of modifications for 11 chromosomal loci by TALENs and 2 by Cas9/gRNAs with different combinations of gRNA pairs, including clusters of miRNA and protein-coding genes. Seven of eight TALEN-targeted lines transmitted the deletions and one transmitted the inversion through germ line. Our findings indicate that both TALENs and Cas9/gRNAs can be used as an efficient tool to engineer genomes to achieve large deletions or inversions, including fragments covering multiple genes and non-coding sequences. To facilitate the analyses and application of existing ZFN, TALEN and CRISPR/Cas data, we have updated our EENdb database to provide a chromosomal view of all reported engineered endonucleases targeting human and zebrafish genomes.
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Transdifferentiation of fast skeletal muscle into functional endothelium in vivo by transcription factor Etv2.
PLoS Biol.
PUBLISHED: 06-01-2013
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Etsrp/Etv2 (Etv2) is an evolutionarily conserved master regulator of vascular development in vertebrates. Etv2 deficiency prevents the proper specification of the endothelial cell lineage, while its overexpression causes expansion of the endothelial cell lineage in the early embryo or in embryonic stem cells. We hypothesized that Etv2 alone is capable of transdifferentiating later somatic cells into endothelial cells. Using heat shock inducible Etv2 transgenic zebrafish, we demonstrate that Etv2 expression alone is sufficient to transdifferentiate fast skeletal muscle cells into functional blood vessels. Following heat treatment, fast skeletal muscle cells turn on vascular genes and repress muscle genes. Time-lapse imaging clearly shows that muscle cells turn on vascular gene expression, undergo dramatic morphological changes, and integrate into the existing vascular network. Lineage tracing and immunostaining confirm that fast skeletal muscle cells are the source of these newly generated vessels. Microangiography and observed blood flow demonstrated that this new vasculature is capable of supporting circulation. Using pharmacological, transgenic, and morpholino approaches, we further establish that the canonical Wnt pathway is important for induction of the transdifferentiation process, whereas the VEGF pathway provides a maturation signal for the endothelial fate. Additionally, overexpression of Etv2 in mammalian myoblast cells, but not in other cell types examined, induced expression of vascular genes. We have demonstrated in zebrafish that expression of Etv2 alone is sufficient to transdifferentiate fast skeletal muscle into functional endothelial cells in vivo. Given the evolutionarily conserved function of this transcription factor and the responsiveness of mammalian myoblasts to Etv2, it is likely that mammalian muscle cells will respond similarly.
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Role of factor H binding protein in Neisseria meningitidis virulence and its potential as a vaccine candidate to broadly protect against meningococcal disease.
Microbiol. Mol. Biol. Rev.
PUBLISHED: 05-24-2013
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Neisseria meningitidis is a Gram-negative microorganism that exists exclusively in humans and can cause devastating invasive disease. Although capsular polysaccharide-based vaccines against serogroups A, C, Y, and W135 are widely available, the pathway to a broadly protective vaccine against serogroup B has been more complex. The last 11 years has seen the discovery and development of the N. meningitidis serogroup B (MnB) outer membrane protein factor H binding protein (fHBP) as a vaccine component. Since the initial discovery of fHBP, a tremendous amount of work has accumulated on the diversity, structure, and regulation of this important protein. fHBP has proved to be a virulence factor for N. meningitidis and a target for functional bactericidal antibodies. fHBP is critical for survival of meningococci in the human host, as it is responsible for the primary interaction with human factor H (fH). Binding of hfH by the meningococcus serves to downregulate the host alternative complement pathway and helps the organism evade host innate immunity. Preclinical studies have shown that an fHBP-based vaccine can elicit serum bactericidal antibodies capable of killing MnB, and the vaccine has shown very encouraging results in human clinical trials. This report reviews our current knowledge of fHBP. In particular, we discuss the recent advances in our understanding of fHBP, its importance to N. meningitidis, and its potential role as a vaccine for preventing MnB disease.
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Inactivation of mTORC1 in the developing brain causes microcephaly and affects gliogenesis.
J. Neurosci.
PUBLISHED: 05-03-2013
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The mammalian target of rapamycin (mTOR) regulates cell growth in response to various intracellular and extracellular signals. It assembles into two multiprotein complexes: the rapamycin-sensitive mTOR complex 1 (mTORC1) and the rapamycin-insensitive mTORC2. In this study, we inactivated mTORC1 in mice by deleting the gene encoding raptor in the progenitors of the developing CNS. Mice are born but never feed and die within a few hours. The brains deficient for raptor show a microcephaly starting at E17.5 that is the consequence of a reduced cell number and cell size. Changes in cell cycle length during late cortical development and increased cell death both contribute to the reduction in cell number. Neurospheres derived from raptor-deficient brains are smaller, and differentiation of neural progenitors into glia but not into neurons is inhibited. The differentiation defect is paralleled by decreased Stat3 signaling, which is a target of mTORC1 and has been implicated in gliogenesis. Together, our results show that postnatal survival, overall brain growth, and specific aspects of brain development critically depend on mTORC1 function.
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Fev regulates hematopoietic stem cell development via ERK signaling.
Blood
PUBLISHED: 04-16-2013
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Reprogramming of somatic cells to desired cell types holds great promise in regenerative medicine. However, production of transplantable hematopoietic stem cells (HSCs) in vitro by defined factors has not yet been achieved. Therefore, it is critical to fully understand the molecular mechanisms of HSC development in vivo. Here, we show that Fev, an ETS transcription factor, is a pivotal regulator of HSC development in vertebrates. In fev-deficient zebrafish embryos, the first definitive HSC population was compromised and fewer T cells were found in the thymus. Genetic and chemical analyses support a mechanism whereby Fev regulates HSC through direct regulation of ERK signaling. Blastula transplant assay demonstrates that Fev regulation of HSC development is cell autonomous. Experiments performed with purified cord blood show that fev is expressed and functions in primitive HSCs in humans, indicating its conserved role in higher vertebrates. Our data indicate that Fev-ERK signaling is essential for hemogenic endothelium-based HSC development.
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Essential role of SH3-domain GRB2-like 3 for vascular lumen maintenance in zebrafish.
Arterioscler. Thromb. Vasc. Biol.
PUBLISHED: 03-28-2013
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Studying the underlying molecular mechanisms for maintaining stereotyped vascular lumen diameters should help toward a comprehensive understanding of vascular homeostasis and function. We aimed to determine the role of SH3-domain GRB2-like 3 (Sh3gl3) and its interacting pathways in dorsal aorta (DA) maintenance in zebrafish.
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Sustained activation of mTORC1 in skeletal muscle inhibits constitutive and starvation-induced autophagy and causes a severe, late-onset myopathy.
Cell Metab.
PUBLISHED: 03-21-2013
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Autophagy is a catabolic process that ensures homeostatic cell clearance and is deregulated in a growing number of myopathological conditions. Although FoxO3 was shown to promote the expression of autophagy-related genes in skeletal muscle, the mechanisms triggering autophagy are unclear. We show that TSC1-deficient mice (TSCmKO), characterized by sustained activation of mTORC1, develop a late-onset myopathy related to impaired autophagy. In young TSCmKO mice, constitutive and starvation-induced autophagy is blocked at the induction steps via mTORC1-mediated inhibition of Ulk1, despite FoxO3 activation. Rapamycin is sufficient to restore autophagy in TSCmKO mice and improves the muscle phenotype of old mutant mice. Inversely, abrogation of mTORC1 signaling by depletion of raptor induces autophagy regardless of FoxO inhibition. Thus, mTORC1 is the dominant regulator of autophagy induction in skeletal muscle and ensures a tight coordination of metabolic pathways. These findings may open interesting avenues for therapeutic strategies directed toward autophagy-related muscle diseases.
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Clinician impression versus prescription drug monitoring program criteria in the assessment of drug-seeking behavior in the emergency department.
Ann Emerg Med
PUBLISHED: 03-11-2013
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We compare emergency provider impression of drug-seeking behavior with objective criteria from a state prescription drug monitoring program, assess change in opioid pain reliever prescribing after prescription drug monitoring program review, and examine clinical factors associated with suspected drug-seeking behavior.
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Glycated haemoglobin A1 c for diagnosing diabetes in Chinese subjects over 50 years old: a community-based cross-sectional study.
Clin. Endocrinol. (Oxf)
PUBLISHED: 03-07-2013
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OBJECTIVE: Little is known about using glycated haemoglobin A1 c (HbA1 c) to diagnose diabetes in Chinese subjects over 50 years old. This study aims to evaluate HbA1 c in diagnosing diabetes and identify the optimal threshold to be used in Chinese community subjects aged over 50 years. METHODS: A community-based cross-sectional survey was conducted from October 2010 to January 2011 in Shipai community of Guangzhou, Guangdong, China. A total of 1494 subjects (72·8%) aged over 50 years were investigated. Fasting plasma glucose (FPG1st ) and HbA1 c were assayed in each participant. Diabetic candidates with FPG1st  ? 5·6 mmol/l or HbA1 c ? 39 mmol/mol (5·7%) were informed to undergo a 75-g oral glucose tolerance test (OGTT). Diagnosis of diabetes was made by 1999 World Health Organization criteria. Sensitivity and specificity of HbA1 c for diagnosing diabetes were calculated by receiver operating characteristics (ROC) curve. RESULTS: Among 1494 subjects, 161 subjects (10·8%) with previously diagnosed diabetes and 21 with missing data were excluded. Among the remaining 1312 subjects (87·8%), 861 subjects (65·6%) with either FPG1st  ? 5·6 mmol/l or HbA1c ? 39 mmol/mol (5·7%) were invited to perform OGTT. Finally, 453 subjects (52·6%) performed OGTT (FPG2nd and 2-h plasma glucose were measured) and 54 subjects (11·9%) were identified as being diabetes. The area under ROC curve was 0·916 (0·887-0·940) for HbA1 c and 0·972 (0·953-0·985) for FPG2nd in diagnosing diabetes (P = 0·045). An HbA1 c threshold of 48 mmol/mol (6·5%) yielded the highest combination of sensitivity (75·9%) and specificity (95·5%) for diagnosing diabetes. CONCLUSION: An HbA1 c threshold of 48 mmol/mol (6·5%) was highly specific and had a good sensitivity for diagnosing diabetes among Chinese subjects aged over 50 years with FPG ? 5·6 mmol/l or HbA1 c ? 39 mmol/mol (5·7%). This threshold may be suitable for diagnosing diabetes in Chinese subjects over 50 years old.
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One-pot syntheses of isoquinolin-3-ones and benzo-1,4-diazepin-2,5-diones utilizing Ugi-4CR post-transformation strategy.
ACS Comb Sci
PUBLISHED: 03-07-2013
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One-pot and efficient syntheses of structurally diverse isoquinolin-3-ones and isoquinolin-3-one-based benzo-1,4-diazepin-2,5-diones have been developed. The notable features of the process include the Ugi condensation of monomasked phthalaldehydes with amines, carboxylic acids, and isonitriles, followed by HClO4-mediated intramolecular condensation of the carbonyl with amide.
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Eif3ba regulates cranial neural crest development by modulating p53 in zebrafish.
Dev. Biol.
PUBLISHED: 03-06-2013
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Congenital diseases caused by abnormal development of the cranial neural crest usually present craniofacial malformations and heart defects while the precise mechanism is not fully understood. Here, we show that the zebrafish eif3ba mutant caused by pseudo-typed retrovirus insertion exhibited a similar phenotype due to the hypogenesis of cranial neural crest cells (NCCs). The derivatives of cranial NCCs, including the NCC-derived cell population of pharyngeal arches, craniofacial cartilage, pigment cells and the myocardium derived from cardiac NCCs, were affected in this mutant. The expression of several neural crest marker genes, including crestin, dlx2a and nrp2b, was specifically reduced in the cranial regions of the eif3ba mutant. Through fluorescence-tracing of the cranial NCC migration marker nrp2b, we observed reduced intensity of NCC-derived cells in the heart. In addition, p53 was markedly up-regulated in the eif3ba mutant embryos, which correlated with pronounced apoptosis in the cranial area as shown by TUNEL staining. These findings suggest a novel function of eif3ba during embryonic development and a novel level of regulation in the process of cranial NCC development, in addition to providing a potential animal model to mimic congenital diseases due to cranial NCC defects. Furthermore, we report the identification of a novel transgenic fish line Et(gata2a:EGFP)pku418 to trace the migration of cranial NCCs (including cardiac NCCs); this may serve as an invaluable tool for investigating the development and dynamics of cranial NCCs during zebrafish embryogenesis.
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A novel PEGylated liposome-encapsulated SANT75 suppresses tumor growth through inhibiting hedgehog signaling pathway.
PLoS ONE
PUBLISHED: 02-24-2013
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The Hedgehog (Hh) pathway inhibitors have shown great promise in cancer therapeutics. SANT75, a novel compound we previously designed to specially inhibit the Smoothened (SMO) protein in the Hh pathway, has greater inhibitory potency than many of commonly used Hh inhibitors. However, preclinical studies of SANT75 revealed water insolubility and acute toxicity. To overcome these limitations, we developed a liposomal formulation of SANT75 and investigated its antitumor efficacy in vitro and in vivo. We encapsulated SANT75 into PEGylated liposome and the mean particle size distribution and zeta-potential (ZP) of liposomes were optimized. Using the Shh-light2 cell and Gli-GFP or Flk-GFP transgenic reporter zebrafish, we confirmed that liposome-encapsulated SANT75 inhibited Hh activity with similar potency as the original SANT75. SANT75 encapsulated into liposome exerted strong tumor growth-inhibiting effects in vitro and in vivo. In addition, the liposomal SANT75 therapy efficiently improved the survival time of tumor-bearing mice without obvious systemic toxicity. The pathological morphology and immunohistochemistry staining revealed that liposomal SANT75 induced tumor cell apoptosis, inhibited tumor angiogenesis as assessed by CD31 and down-regulated the expression of Hh target protein Gli-1 in tumor tissues. Our findings suggest that liposomal formulated SANT75 has improved solubility and bioavailability and should be further developed as a drug candidate for treating tumors with abnormally high Hh activity.
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Differential response of skeletal muscles to mTORC1 signaling during atrophy and hypertrophy.
Skelet Muscle
PUBLISHED: 02-15-2013
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Skeletal muscle mass is determined by the balance between protein synthesis and degradation. Mammalian target of rapamycin complex 1 (mTORC1) is a master regulator of protein translation and has been implicated in the control of muscle mass. Inactivation of mTORC1 by skeletal muscle-specific deletion of its obligatory component raptor results in smaller muscles and a lethal dystrophy. Moreover, raptor-deficient muscles are less oxidative through changes in the expression PGC-1?, a critical determinant of mitochondrial biogenesis. These results suggest that activation of mTORC1 might be beneficial to skeletal muscle by providing resistance to muscle atrophy and increasing oxidative function. Here, we tested this hypothesis by deletion of the mTORC1 inhibitor tuberous sclerosis complex (TSC) in muscle fibers.
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A large-scale zebrafish gene knockout resource for the genome-wide study of gene function.
Genome Res.
PUBLISHED: 02-04-2013
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With the completion of the zebrafish genome sequencing project, it becomes possible to analyze the function of zebrafish genes in a systematic way. The first step in such an analysis is to inactivate each protein-coding gene by targeted or random mutation. Here we describe a streamlined pipeline using proviral insertions coupled with high-throughput sequencing and mapping technologies to widely mutagenize genes in the zebrafish genome. We also report the first 6144 mutagenized and archived F1s predicted to carry up to 3776 mutations in annotated genes. Using in vitro fertilization, we have rescued and characterized ~0.5% of the predicted mutations, showing mutation efficacy and a variety of phenotypes relevant to both developmental processes and human genetic diseases. Mutagenized fish lines are being made freely available to the public through the Zebrafish International Resource Center. These fish lines establish an important milestone for zebrafish genetics research and should greatly facilitate systematic functional studies of the vertebrate genome.
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TALEN-mediated precise genome modification by homologous recombination in zebrafish.
Nat. Methods
PUBLISHED: 01-15-2013
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We report gene targeting via homologous recombination in zebrafish. We co-injected fertilized eggs with transcription activator-like effector nuclease mRNAs and a donor vector with long homologous arms targeting the tyrosine hydroxylase (th) locus, and we observed effective gene modification that was transmitted through the germ line. We also successfully targeted two additional genes. Homologous recombination in zebrafish with a dsDNA donor expands the utility of this model organism.
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A novel anti-tumor inhibitor identified by virtual screen with PLK1 structure and zebrafish assay.
PLoS ONE
PUBLISHED: 01-01-2013
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Polo-like kinase 1 (PLK1), one of the key regulators of mitosis, is a target for cancer therapy due to its abnormally high activity in several tumors. Plk1 is highly conserved and shares a nearly identical 3-D structure between zebrafish and humans. The initial 10 mitoses of zebrafish embryonic cleavages occur every?30 minutes, and therefore provide a rapid assay to evaluate mitosis inhibitors including those targeting Plk1. To increase efficiency and specificity, we first performed a computational virtual screen of?60000 compounds against the human Plk1 3-D structure docked to both its kinase and Polo box domain. 370 candidates with the top free-energy scores were subjected to zebrafish assay and 3 were shown to inhibit cell division. Compared to general screen for compounds inhibiting zebrafish embryonic cleavage, computation increased the efficiency by 11 folds. One of the 3 compounds, named I2, was further demonstrated to effectively inhibit multiple tumor cell proliferation in vitro and PC3 prostate cancer growth in Xenograft mouse model in vivo. Furthermore, I2 inhibited Plk1 enzyme activity in a dose dependent manner. The IC50 values of I2 in these assays are compatible to those of ON-01910, a Plk1 inhibitor currently in Phase III clinic trials. Our studies demonstrate that zebrafish assays coupled with computational screening significantly improves the efficiency of identifying specific regulators of biological targets. The PLK1 inhibitor I2, and its analogs, may have potential in cancer therapeutics.
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Myopathy caused by mammalian target of rapamycin complex 1 (mTORC1) inactivation is not reversed by restoring mitochondrial function.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 12-05-2011
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Mammalian target of rapamycin complex 1 (mTORC1) is central to the control of cell, organ, and body size. Skeletal muscle-specific inactivation of mTORC1 in mice results in smaller muscle fibers, fewer mitochondria, increased glycogen stores, and a progressive myopathy that causes premature death. In mTORC1-deficient muscles, peroxisome proliferator-activated receptor gamma coactivator 1-? (PGC-1?), which regulates mitochondrial biogenesis and glucose homeostasis, is strongly down-regulated. Here we tested whether induction of mitochondrial biogenesis pharmacologically or by the overexpression of PGC-1? is sufficient to reverse the phenotype of mice deficient for mTORC1. We show that both approaches normalize mitochondrial function, such as oxidative capacity and expression of mitochondrial genes. However, they do not prevent or delay the progressive myopathy. In addition, we find that mTORC1 has a much stronger effect than PGC-1? on the glycogen content in muscle. This effect is based on the strong activation of PKB/Akt in mTORC1-deficient mice. We also show that activation of PKB/Akt not only affects glycogen synthesis but also diminishes glycogen degradation. Thus, our work provides strong functional evidence that mitochondrial dysfunction in mice with inactivated mTORC1 signaling is caused by the down-regulation of PGC-1?. However, our data also show that the impairment of mitochondria does not lead directly to the lethal myopathy.
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Etsrp/Etv2 is directly regulated by Foxc1a/b in the zebrafish angioblast.
Circ. Res.
PUBLISHED: 12-01-2011
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Endothelial cells are developmentally derived from angioblasts specified in the mesodermal germ cell layer. The transcription factor etsrp/etv2 is at the top of the known genetic hierarchy for angioblast development. The transcriptional events that induce etsrp expression and angioblast specification are not well understood.
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[Progress in zinc finger nuclease engineering for targeted genome modification].
Yi Chuan
PUBLISHED: 11-05-2011
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Zinc finger nuclease (ZFN) is an artificially engineered hybrid protein that contains a zinc finger protein (ZFP) domain and a Fok I endonuclease cleavage domain. It has recently emerged as a powerful molecular tool for targeted genome modifications. ZFNs recognize and bind to specific DNA sequences to generate a double-strand break (DSB) by its nuclease activity. Based on this finding, various genetic methods, including gene targeting (gene disruption), gene addition, gene correction etc., are being designed to manipulate the genomes of different species at specific loci. One particular advantage of this new technique is its broad applications, which can be employed to generate desirable inheritable mutations both at the organismal level and at the cellular level. Here, we review the recent progress and prospects of ZFN technology. This article focused on the mechanism of how it works, currently available target assessment, ZFP library construction and screening methods, target modification strategies, as well as a collection of specie and genes that have been successfully modified by ZFN. This review will provide a useful reference for researchers who are interested in applying this new technique in their studies.
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Endocrine pancreas development in zebrafish.
Cell Cycle
PUBLISHED: 10-15-2011
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Type 1 diabetes results from the autoimmune destruction of insulin-producing pancreatic ? cells. Current efforts to cure diabetes are aimed at replenishing damaged cells by generating a new supply of ? cells in vitro. The most promising strategy for achieving this goal is to differentiate embryonic stem (ES) cells by sequentially exposing them to signaling molecules that they would normally encounter in vivo. This approach requires a thorough understanding of the temporal sequence of the signaling events underlying pancreatic ?-cell induction during embryonic development. The zebrafish system has emerged as a powerful tool in the study of pancreas development. In this review, we provide a temporal summary of pancreas development in zebrafish with a special focus on the formation of pancreatic ? cells.
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Genetic suppressor screens in haploids.
Methods Cell Biol.
PUBLISHED: 09-20-2011
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As a vertebrate genetic model, the zebrafish has been well recognized for its strength in studying a variety of biological processes and human diseases. Traditional forward genetic screens in zebrafish have generated a large pool of mutants with interesting phenotypes resembling human diseases but the underlying mechanisms are not well understood. A powerful approach to elucidate the mechanisms of these mutants is the modifier screen, which identifies 2(nd)-site mutations that specifically enhance or block the phenotype of a given mutant. Here we described the first genetic suppressor screen in zebrafish, which identifies a novel transcriptional mechanism regulating erythropoiesis. In combination with the haploid genetics in zebrafish, we have shown the feasibility and strength of a modifier screen in zebrafish. This strategy will greatly broaden the utility of the zebrafish as a model for making original discoveries and establishing novel paradigms for understanding vertebrate biology.
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High content screening in zebrafish speeds up hazard ranking of transition metal oxide nanoparticles.
ACS Nano
PUBLISHED: 08-25-2011
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Zebrafish is an aquatic organism that can be used for high content safety screening of engineered nanomaterials (ENMs). We demonstrate, for the first time, the use of high content bright-field and fluorescence-based imaging to compare the toxicological effect of transition metal oxide (CuO, ZnO, NiO, and Co(3)O(4)) nanoparticles in zebrafish embryos and larvae. High content bright-field imaging demonstrated potent and dose-dependent hatching interference in the embryos, with the exception of Co(3)O(4) which was relatively inert. We propose that the hatching interference was due to the shedding of Cu and Ni ions, compromising the activity of the hatching enzyme, ZHE1, similar to what we previously proposed for Zn(2+). This hypothesis is based on the presence of metal-sensitive histidines in the catalytic center of this enzyme. Co-introduction of a metal ion chelator, diethylene triamine pentaacetic acid (DTPA), reversed the hatching interference of Cu, Zn, and Ni. While neither the embryos nor larvae demonstrated morphological abnormalities, high content fluorescence-based imaging demonstrated that CuO, ZnO, and NiO could induce increased expression of the heat shock protein 70:enhanced green fluorescence protein (hsp70:eGFP) in transgenic zebrafish larvae. Induction of this response by CuO required a higher nanoparticle dose than the amount leading to hatching interference. This response was also DTPA-sensitive. We demonstrate that high content imaging of embryo development, morphological abnormalities, and HSP70 expression can be used for hazard ranking and determining the dose-response relationships leading to ENM effects on the development of the zebrafish embryo.
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A SNX10/V-ATPase pathway regulates ciliogenesis in vitro and in vivo.
Cell Res.
PUBLISHED: 08-16-2011
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Sorting nexins (SNXs) are phosphoinositide-binding proteins implicated in the sorting of various membrane proteins in vitro, but the in vivo functions of them remain largely unknown. We reported previously that SNX10 is a unique member of the SNX family genes in that it has vacuolation activity in cells. We investigate the biological function of SNX10 by loss-of-function assay in this study and demonstrate that SNX10 is required for the formation of primary cilia in cultured cells. In zebrafish, SNX10 is involved in ciliogenesis in the Kupffers vesicle and essential for left-right patterning of visceral organs. Mechanistically, SNX10 interacts with V-ATPase complex and targets it to the centrosome where ciliogenesis is initiated. Like SNX10, V-ATPase regulates ciliogenesis in vitro and in vivo and does so synergistically with SNX10. We further discover that SNX10 and V-ATPase regulate the ciliary trafficking of Rab8a, which is a critical regulator of ciliary membrane extension. These results identify an SNX10/V-ATPase-regulated vesicular trafficking pathway that is crucial for ciliogenesis, and reveal that SNX10/V-ATPase, through the regulation of cilia formation in various organs, play an essential role during early embryonic development.
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A novel xenograft model in zebrafish for high-resolution investigating dynamics of neovascularization in tumors.
PLoS ONE
PUBLISHED: 05-14-2011
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Tumor neovascularization is a highly complex process including multiple steps. Understanding this process, especially the initial stage, has been limited by the difficulties of real-time visualizing the neovascularization embedded in tumor tissues in living animal models. In the present study, we have established a xenograft model in zebrafish by implanting mammalian tumor cells into the perivitelline space of 48 hours old Tg(Flk1:EGFP) transgenic zebrafish embryos. With this model, we dynamically visualized the process of tumor neovascularization, with unprecedented high-resolution, including new sprouts from the host vessels and the origination from VEGFR2(+) individual endothelial cells. Moreover, we quantified their contributions during the formation of vascular network in tumor. Real-time observations revealed that angiogenic sprouts in tumors preferred to connect each other to form endothelial loops, and more and more endothelial loops accumulated into the irregular and chaotic vascular network. The over-expression of VEGF165 in tumor cells significantly affected the vascularization in xenografts, not only the number and size of neo-vessels but the abnormalities of tumor vascular architecture. The specific inhibitor of VEGFR2, SU5416, significantly inhibited the vascularization and the growth of melanoma xenografts, but had little affects to normal vessels in zebrafish. Thus, this zebrafish/tumor xenograft model not only provides a unique window to investigate the earliest events of tumoral neoangiogenesis, but is sensitive to be used as an experimental platform to rapidly and visually evaluate functions of angiogenic-related genes. Finally, it also offers an efficient and cost-effective means for the rapid evaluation of anti-angiogenic chemicals.
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Structural and biochemical characterization of Staphylococcus aureus clumping factor B/ligand interactions.
J. Biol. Chem.
PUBLISHED: 05-03-2011
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Clumping factor B (ClfB) from Staphylococcus aureus is a bifunctional protein that binds to human cytokeratin 10 (K10) and fibrinogen (Fg). ClfB has been implicated in S. aureus colonization of nasal epithelium and is therefore a key virulence factor. People colonized with S. aureus are at an increased risk for invasive staphylococcal disease. In this study, we have determined the crystal structures of the ligand-binding region of ClfB in an apo-form and in complex with human K10 and Fg ?-chain-derived peptides, respectively. We have determined the structures of MSCRAMM binding to two ligands with different sequences in the same site showing the versatile nature of the ligand recognition mode of microbial surface components recognizing adhesive matrix molecules. Both ligands bind ClfB by parallel ?-sheet complementation as observed for the clumping factor A·?-chain peptide complex. The ?-sheet complementation is shorter in the ClfB·Fg ?-chain peptide complex. The structures show that several residues in ClfB are important for binding to both ligands, whereas others only make contact with one of the ligands. A common motif GSSGXG found in both ligands is part of the ClfB-binding site. This motif is found in many human proteins thus raising the possibility that ClfB recognizes additional ligands.
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Targeting zebrafish and murine pituitary corticotroph tumors with a cyclin-dependent kinase (CDK) inhibitor.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 05-02-2011
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Cushing disease caused by adrenocorticotropin (ACTH)-secreting pituitary adenomas leads to hypercortisolemia predisposing to diabetes, hypertension, osteoporosis, central obesity, cardiovascular morbidity, and increased mortality. There is no effective pituitary targeted pharmacotherapy for Cushing disease. Here, we generated germline transgenic zebrafish with overexpression of pituitary tumor transforming gene (PTTG/securin) targeted to the adenohypophyseal proopiomelanocortin (POMC) lineage, which recapitulated early features pathognomonic of corticotroph adenomas, including corticotroph expansion and partial glucocorticoid resistance. Adult Tg:Pomc-Pttg fish develop neoplastic coticotrophs and pituitary cyclin E up-regulation, as well as metabolic disturbances mimicking hypercortisolism caused by Cushing disease. Early development of corticotroph pathologies in Tg:Pomc-Pttg embryos facilitated drug testing in vivo. We identified a pharmacologic CDK2/cyclin E inhibitor, R-roscovitine (seliciclib; CYC202), which specifically reversed corticotroph expansion in live Tg:Pomc-Pttg embryos. We further validated that orally administered R-roscovitine suppresses ACTH and corticosterone levels, and also restrained tumor growth in a mouse model of ACTH-secreting pituitary adenomas. Molecular analyses in vitro and in vivo showed that R-roscovitine suppresses ACTH expression, induces corticotroph tumor cell senescence and cell cycle exit by up-regulating p27, p21 and p57, and downregulates cyclin E expression. The results suggest that use of selective CDK inhibitors could effectively target corticotroph tumor growth and hormone secretion.
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Apoptosis inhibitors and mini-agrin have additive benefits in congenital muscular dystrophy mice.
EMBO Mol Med
PUBLISHED: 04-29-2011
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Mutations in LAMA2 cause a severe form of congenital muscular dystrophy, called MDC1A. Studies in mouse models have shown that transgenic expression of a designed, miniaturized form of the extracellular matrix molecule agrin (mini-agrin) or apoptosis inhibition by either overexpression of Bcl2 or application of the pharmacological substance omigapil can ameliorate the disease. Here, we tested whether mini-agrin and anti-apoptotic agents act on different pathways and thus exert additive benefits in MDC1A mouse models. By combining mini-agrin with either transgenic Bcl2 expression or oral omigapil application, we show that the ameliorating effect of mini-agrin, which acts by restoring the mechanical stability of muscle fibres and, thereby, reduces muscle fibre breakdown and concomitant fibrosis, is complemented by apoptosis inhibitors, which prevent the loss of muscle fibres. Treatment of mice with both agents results in improved muscle regeneration and increased force. Our results show that the combination of mini-agrin and anti-apoptosis treatment has beneficial effects that are significantly bigger than the individual treatments and suggest that such a strategy might also be applicable to MDC1A patients.
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Distinct contributions of angiogenesis and vascular co-option during the initiation of primary microtumors and micrometastases.
Carcinogenesis
PUBLISHED: 04-23-2011
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Primary tumors and metastases have been thought to initiate avascularly as multicellular aggregates and later induce angiogenesis or initiate vascularly by co-opting pre-existing host blood vessels without inducing angiogenesis. These two distinct concepts of microtumor vascularization have raised significant controversies. To clarify intratumoral vascularization and tumor cell behaviors at single-cell level during the earliest stage of microtumor initiation, we established primary and metastatic microtumor models in Tg(flk1:EGFP) transgenic zebrafish. We found that tumor cells preferred to initiate avascularly as multicellular aggregates and only later (50-100 cells in size) induced angiogenesis in blood-supply-sufficient microenvironments. In blood-supply-deficient microenvironments, less tumor cells (20-30 cells per fish) managed to co-opt and migrate along host vessels, whereas more tumor cells (100-300 cells per fish) could immediately induce angiogenesis without obvious cell migration. In a metastatic model, we clearly observed that tumor cells co-opted, migrated along and proliferated on the surface of host vessels at an early stage after they extravasated from host vessels and induced angiogenesis later when micromatastases comprised only 15-30 tumor cells. Moreover, the inducement of neovessels accelerated the growth of micromatastases in size, meanwhile, decreased the migration of tumor cells on the surface of host vessels. These results suggest that vessel co-option and angiogenesis have distinct contributions during the initiation of microtumors. Microtumors initiated reasonably through co-opting host vessels or inducing angiogenesis, depending on the differences of local microenvironments and cell numbers in microtumors. The results in this study may have important implications for the therapeutic application of antiangiogenic strategies.
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Wdr18 is required for Kupffers vesicle formation and regulation of body asymmetry in zebrafish.
PLoS ONE
PUBLISHED: 04-11-2011
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Correct specification of the left-right (L-R) axis is important for organ morphogenesis. Conserved mechanisms involving cilia rotation inside node-like structures and asymmetric Nodal signaling in the lateral plate mesoderm (LPM), which are important symmetry-breaking events, have been intensively studied. In zebrafish, the clustering and migration of dorsal forerunner cells (DFCs) is critical for the formation of the Kuppfers vesicle (KV). However, molecular events underlying DFC clustering and migration are less understood. The WD-repeat proteins function in a variety of biological processes, including cytoskeleton assembly, intracellular trafficking, mRNA splicing, transcriptional regulation and cell migration. However, little is known about the function of WD-repeat proteins in L-R asymmetry determination. Here, we report the identification and functional analyses of zebrafish wdr18, a novel gene that encodes a WD-repeat protein that is highly conserved among vertebrate species. wdr18 was identified from a Tol2 transposon-mediated enhancer trap screen. Follow-up analysis of wdr18 mRNA expression showed that it was detected in DFCs or the KV progenitor cells and later in the KV at early somitogenesis stages. Morpholino knockdown of wdr18 resulted in laterality defects in the visceral organs, which were preceded by the mis-expression of Nodal-related genes, including spaw and pitx2. Examination of morphants at earlier stages revealed that the KV had fewer and shorter cilia which are immotile and a smaller cavity. We further investigated the organization of DFCs in wdr18 morphant embryos using ntl and sox17 as specific markers and found that the clustering and migration of DFC was altered, leading to a disorganized KV. Finally, through a combination of wdr18 and itgb1b morpholino injections, we provided evidence that wdr18 and itgb1b genetically interact in the laterality determination process. Thus, we reveal a new and essential role for WD-repeat proteins in the determination and regulation of L-R asymmetry and propose a potential mechanism for wdr18 in the regulation of DFC clustering and migration and KV formation.
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Combinatory action of VEGFR2 and MAP kinase pathways maintains endothelial-cell integrity.
Cell Res.
PUBLISHED: 03-22-2011
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Blood vessels normally maintain stereotyped lumen diameters and their stable structures are crucial for vascular function. However, very little is known about the molecular mechanisms controlling the maintenance of vessel diameters and the integrity of endothelial cells. We investigated this issue in zebrafish embryos by a chemical genetics approach. Small molecule libraries were screened using live Tg(kdrl:GRCFP)(zn1) transgenic embryos in which endothelial cells are specifically labeled with GFP. By analyzing the effects of compounds on the morphology and function of embryonic blood vessels after lumen formation, PP1, a putative Src kinase inhibitor, was identified as capable of specifically reducing vascular lumen size by interrupting endothelial-cell integrity. The inhibitory effect is not due to Src or general VEGF signaling inhibition because another Src inhibitor and Src morpholino as well as several VEGFR inhibitors failed to produce a similar phenotype. After profiling a panel of 22 representative mammalian kinases and surveying published data, we selected a few possible new candidates. Combinational analysis of these candidate kinase inhibitors established that PP1 induced endothelial collapse by inhibiting both the VEGFR2 and MAP kinase pathways. More importantly, combinatory use of two clinically approved drugs Dasatinib and Sunitinib produced the same phenotype. This is the first study to elucidate the pathways controlling maintenance of endothelial integrity using a chemical genetics approach, indicating that endothelial integrity is controlled by the combined action of the VEGFR2 and MAP kinase pathways. Our results also suggest the possible side effect of the combination of two anticancer drugs on the circulatory system.
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Muscle-selective synaptic disassembly and reorganization in MuSK antibody positive MG mice.
Exp. Neurol.
PUBLISHED: 02-23-2011
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MuSK antibody seropositive (MuSK+) Myasthenia Gravis (MG) patients present a distinct selective fatigue, and sometimes atrophy, of bulbar, facial and neck muscles. Here, we study the mechanism underlying the focal muscle involvement in mice with MuSK+ experimental autoimmune MG (EAMG). 8 week-old female wildtype C57BL6 mice and transgenic mice, which express yellow fluorescence protein (YFP) in their motor neurons, were immunized with the extracellular domain of rat MuSK and compared with control mice. The soleus, EDL, sternomastoid, omohyoid, thoracic paraspinal and masseter muscles were examined for pre- and postsynaptic changes with whole mount immunostaining and confocal microscopy. Neuromuscular junction derangement was quantified and compared between muscles and correlated with transcript levels of MuSK and other postsynaptic genes. Correlating with the EAMG disease grade, the postsynaptic acetylcholine receptor (AChR) clusters were severely fragmented with a subsequent reduction also of the presynaptic nerve terminal area. Among the muscles analyzed, the thoracic paraspinal, sternomastoid and masseter muscles were more affected than the leg muscles. The masseter muscle was the most affected, leading to denervation and atrophy and this severity correlated with the lowest levels of MuSK mRNA. On the contrary, the soleus with high MuSK mRNA levels had less postsynaptic perturbation and more terminal nerve sprouting. We propose that low muscle-intrinsic MuSK levels render some muscles, such as the masseter, more vulnerable to the postsynaptic perturbation of MuSK antibodies with subsequent denervation and atrophy. These findings augment our understanding of the sometimes severe, facio-bulbar phenotype of MuSK+ MG.
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Use of a high-throughput screening approach coupled with in vivo zebrafish embryo screening to develop hazard ranking for engineered nanomaterials.
ACS Nano
PUBLISHED: 02-16-2011
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Because of concerns about the safety of a growing number of engineered nanomaterials (ENM), it is necessary to develop high-throughput screening and in silico data transformation tools that can speed up in vitro hazard ranking. Here, we report the use of a multiparametric, automated screening assay that incorporates sublethal and lethal cellular injury responses to perform high-throughput analysis of a batch of commercial metal/metal oxide nanoparticles (NP) with the inclusion of a quantum dot (QD1). The responses chosen for tracking cellular injury through automated epifluorescence microscopy included ROS production, intracellular calcium flux, mitochondrial depolarization, and plasma membrane permeability. The z-score transformed high volume data set was used to construct heat maps for in vitro hazard ranking as well as showing the similarity patterns of NPs and response parameters through the use of self-organizing maps (SOM). Among the materials analyzed, QD1 and nano-ZnO showed the most prominent lethality, while Pt, Ag, SiO2, Al2O3, and Au triggered sublethal effects but without cytotoxicity. In order to compare the in vitro with the in vivo response outcomes in zebrafish embryos, NPs were used to assess their impact on mortality rate, hatching rate, cardiac rate, and morphological defects. While QDs, ZnO, and Ag induced morphological abnormalities or interfered in embryo hatching, Pt and Ag exerted inhibitory effects on cardiac rate. Ag toxicity in zebrafish differed from the in vitro results, which is congruent with this materials designation as extremely dangerous in the environment. Interestingly, while toxicity in the initially selected QD formulation was due to a solvent (toluene), supplementary testing of additional QDs selections yielded in vitro hazard profiling that reflect the release of chalcogenides. In conclusion, the use of a high-throughput screening, in silico data handling and zebrafish testing may constitute a paradigm for rapid and integrated ENM toxicological screening.
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Chemical screening with zebrafish embryos.
Methods Mol. Biol.
PUBLISHED: 02-15-2011
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Functional chemicals are very useful tools for molecular biology studies. Due to its small size, large progeny clutch, and embryonic transparency, zebrafish serves as a superb in vivo animal model for chemical compound screens and characterization. During zebrafish embryogenesis, multiple developmental phenotypes can be easily examined under the microscope, therefore allowing a more comprehensive evaluation for identifying novel functional chemicals than cell-based assays. Ever since the first zebrafish-based chemical screen was conducted in the year 2000, many functional chemicals have been discovered using this strategy. In this chapter, we describe how to perform a typical zebrafish-based chemical screen and discuss the details of the protocol by using the example of the identification and characterization of two new Smo inhibitors with a Gli:GFP transgenic line.
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Disruption of LRRK2 does not cause specific loss of dopaminergic neurons in zebrafish.
PLoS ONE
PUBLISHED: 02-01-2011
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Mutations in LRRK2 are genetically linked to Parkinsons disease (PD) but its normal biological function is largely unknown. Sheng et al. recently reported that deletion of the WD40 domain of LRRK2 in zebrafish specifically causes PD-like loss of neurons and behavior defect. However, our similar early study and recent confirming experiments using the same reagents reported by Sheng et al. failed to reproduce the phenotype of the loss of dopaminergic neurons, although the mRNA of LRRK2 was molecularly disrupted. Our study suggests that function of LRRK2 and its usefulness to generate zebrafish PD model needs further evaluation.
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MuSK levels differ between adult skeletal muscles and influence postsynaptic plasticity.
Eur. J. Neurosci.
PUBLISHED: 01-24-2011
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Muscle-specific tyrosine kinase (MuSK) is involved in the formation and maintenance of the neuromuscular junction (NMJ), and is necessary for NMJ integrity. As muscle involvement is strikingly selective in pathological conditions in which MuSK is targeted, including congenital myasthenic syndrome with MuSK mutation and MuSK antibody-seropositive myasthenia gravis, we hypothesized that the postsynaptic response to MuSK-agrin signalling differs between adult muscles. Transcript levels of postsynaptic proteins were compared between different muscles in wild-type adult mice. MuSK expression was high in the soleus and sternomastoid muscles and low in the extensor digitorum longus (EDL) and omohyoid muscles. The acetylcholine receptor (AChR) ? subunit followed a similar expression pattern, whereas expression of Dok-7, Lrp4 and rapsyn was comparable between the muscles. We subsequently examined muscles in mice that overexpressed a miniaturized form of neural agrin or MuSK. In these transgenic mice, the soleus and sternomastoid muscles responded with formation of ectopic AChR clusters, whereas such clusters were almost absent in the EDL and omohyoid muscles. Electroporation of Dok-7 revealed its important role as an activator of MuSK in AChR cluster formation in adult muscles. Together, our findings indicate for the first time that adult skeletal muscles harbour different endogenous levels of MuSK and that these levels determine the ability to form ectopic AChR clusters upon overexpression of agrin or MuSK. We believe that these findings are important for our understanding of adult muscle plasticity and the selective muscle involvement in neuromuscular disorders in which MuSK is diminished.
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Decreased dissolution of ZnO by iron doping yields nanoparticles with reduced toxicity in the rodent lung and zebrafish embryos.
ACS Nano
PUBLISHED: 01-20-2011
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We have recently shown that the dissolution of ZnO nanoparticles and Zn(2+) shedding leads to a series of sublethal and lethal toxicological responses at the cellular level that can be alleviated by iron doping. Iron doping changes the particle matrix and slows the rate of particle dissolution. To determine whether iron doping of ZnO also leads to lesser toxic effects in vivo, toxicity studies were performed in rodent and zebrafish models. First, we synthesized a fresh batch of ZnO nanoparticles doped with 1-10 wt % of Fe. These particles were extensively characterized to confirm their doping status, reduced rate of dissolution in an exposure medium, and reduced toxicity in a cellular screen. Subsequent studies compared the effects of undoped to doped particles in the rat lung, mouse lung, and the zebrafish embryo. The zebrafish studies looked at embryo hatching and mortality rates as well as the generation of morphological defects, while the endpoints in the rodent lung included an assessment of inflammatory cell infiltrates, LDH release, and cytokine levels in the bronchoalveolar lavage fluid. Iron doping, similar to the effect of the metal chelator, DTPA, interfered in the inhibitory effects of Zn(2+) on zebrafish hatching. In the oropharyngeal aspiration model in the mouse, iron doping was associated with decreased polymorphonuclear cell counts and IL-6 mRNA production. Doped particles also elicited decreased heme oxygenase 1 expression in the murine lung. In the intratracheal instillation studies in the rat, Fe doping was associated with decreased polymorphonuclear cell counts, LDH, and albumin levels. All considered, the above data show that Fe doping is a possible safe design strategy for preventing ZnO toxicity in animals and the environment.
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Zebrafish usp39 mutation leads to rb1 mRNA splicing defect and pituitary lineage expansion.
PLoS Genet.
PUBLISHED: 01-13-2011
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Loss of retinoblastoma (Rb) tumor suppressor function is associated with human malignancies. Molecular and genetic mechanisms responsible for tumorigenic Rb downregulation are not fully defined. Through a forward genetic screen and positional cloning, we identified and characterized a zebrafish ubiquitin specific peptidase 39 (usp39) mutation, the yeast and human homolog of which encodes a component of RNA splicing machinery. Zebrafish usp39 mutants exhibit microcephaly and adenohypophyseal cell lineage expansion without apparent changes in major hypothalamic hormonal and regulatory signals. Gene expression profiling of usp39 mutants revealed decreased rb1 and increased e2f4, rbl2 (p130), and cdkn1a (p21) expression. Rb1 mRNA overexpression, or antisense morpholino knockdown of e2f4, partially reversed embryonic pituitary expansion in usp39 mutants. Analysis of pre-mRNA splicing status of critical cell cycle regulators showed misspliced Rb1 pre-mRNA resulting in a premature stop codon. These studies unravel a novel mechanism for rb1 regulation by a neuronal mRNA splicing factor, usp39. Zebrafish usp39 regulates embryonic pituitary homeostasis by targeting rb1 and e2f4 expression, respectively, contributing to increased adenohypophyseal sensitivity to these altered cell cycle regulators. These results provide a mechanism for dysregulated rb1 and e2f4 pathways that may result in pituitary tumorigenesis.
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Antagonistic interactions of hedgehog, Bmp and retinoic acid signals control zebrafish endocrine pancreas development.
Development
PUBLISHED: 01-12-2011
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Pancreatic organogenesis is promoted or restricted by different signaling pathways. In amniotes, inhibition of hedgehog (Hh) activity in the early embryonic endoderm is a prerequisite for pancreatic specification. However, in zebrafish, loss of Hh signaling leads to a severe reduction of ?-cells, leading to some ambiguity as to the role of Hh during pancreas development and whether its function has completely diverged between species. Here, we have employed genetic and pharmacological manipulations to temporally delineate the role of Hh in zebrafish endocrine pancreas development and investigate its relationship with the Bmp and retinoic acid (RA) signaling pathways. We found that Hh is required at the start of gastrulation for the medial migration and differentiation of pdx1-expressing pancreatic progenitors at later stages. This early positive role of Hh promotes ?-cell lineage differentiation by restricting the repressive effects of Bmp. Inhibition of Bmp signaling in the early gastrula leads to increased ?-cell numbers and partially rescued ?-cell formation in Hh-deficient embryos. By the end of gastrulation, Hh switches to a negative role by antagonizing RA-mediated specification of the endocrine pancreas, but continues to promote differentiation of exocrine progenitors. We show that RA downregulates the Hh signaling components ptc1 and smo in endodermal explants, indicating a possible molecular mechanism for blocking axial mesoderm-derived Hh ligands from the prepancreatic endoderm during the specification stage. These results identify multiple sequential roles for Hh in pancreas development and highlight an unexpected antagonistic relationship between Hh and other signaling pathways to control pancreatic specification and differentiation.
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Challenges for the evaluation of Staphylococcus aureus protein based vaccines: monitoring antigenic diversity.
Hum Vaccin
PUBLISHED: 01-01-2011
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Clumping factors A (ClfA) and B (ClfB) are Staphylococcus aureus virulence proteins that are displayed on the cell surface of the organism and have potential as vaccine antigens for the prevention of S. aureus disease. Here we evaluate the phylogeny of S. aureus in the context of antigenic variation of these two surface proteins. ClfA and ClfB gene sequences, along with epidemiological markers (MLST, spa and capsule genotype) were obtained for 224 S. aureus isolates including both historical strains and a collection representative of current MRSA isolates from the United States. Variation within ClfA and ClfB was consistent with the established population biology of S. aureus, namely, that S. aureus strains belong to a relatively small number of clonal lineages, with evolution proceeding mainly by mutation and with little to no recombination between clades. Thus most variation in ClfA and ClfB occurs between but not within lineages, and particular groups of ClfA and ClfB variants are closely linked. This has important implications for vaccine development and assessment as it suggests that a relatively small survey of strains will be representative of the total population variation, whereas for species that evolve mainly by recombination, such as Neisseria meningitidis, analysis of a much larger number of strains is needed to accomplish the same purpose. Our study also revealed evidence for the de-evolution of ClfB and therefore its reduced suitability as a target for vaccine development compared to ClfA.
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Ribosomal protein L11 mutation in zebrafish leads to haematopoietic and metabolic defects.
Br. J. Haematol.
PUBLISHED: 11-29-2010
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Mutations in ribosomal proteins are associated with a congenital syndrome, Diamond-Blackfan anaemia (DBA), manifested by red blood cell aplasia, developmental abnormalities and increased risk of malignancy. Recent studies suggest the involvement of p53 activation in DBA. However, which pathways are involved and how they contribute to the DBA phenotype remains unknown. Here we show that a zebrafish mutant for the rpl11 gene had defects both in the development of haematopoietic stem cells (HSCs) and maintenance of erythroid cells. The molecular signature of the mutant included upregulation of p53 target genes and global changes in metabolism. The changes in several pathways may affect haematopoiesis including upregulation of pro-apoptotic and cell cycle arrest genes, suppression of glycolysis, downregulation of biosynthesis and dysregulation of cytoskeleton. Each of these pathways has been individually implicated in haematological diseases. Inhibition of p53 partially rescued haematopoiesis in the mutant. Altogether, we propose that the unique phenotype of DBA is a sum of several abnormally regulated molecular pathways, mediated by the p53 protein family and p53-independent, which have synergistic impact on haematological and other cellular pathways affected in DBA. Our results provide new insights into the pathogenesis of DBA and point to the potential avenues for therapeutic intervention.
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Stator design of a new type of spherical piezoelectric motor.
IEEE Trans Ultrason Ferroelectr Freq Control
PUBLISHED: 10-05-2010
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The stator design of a new type of spherical motor driven by piezoelectric actuators is developed. A curved piezoelectric actuator is designed to attach to the spherical surface. A series of the curved piezoelectric actuators is laid in a line around a spherical surface. By applying an appropriate voltage signal with phase difference on neighboring actuators, a traveling wave is generated on the hemispherical shell. Each set of curved piezoelectric actuators is designed to provide motion with a single degree-of-freedom (DOF). With two or three sets of the piezoelectric actuators constructed to be mutually perpendicular, the motor can provide 2-DOF or 3-DOF motion. Stator design and analysis and experiment for the 1-, 2-, and 3-DOF conditions are presented in this article. Analytical calculation and experiment results of several fundamental characteristics of the stator are in good agreement. Performance evaluation of rotation speed and torque of the stator and some implementation problems are also addressed.
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ff1b, the SF1 ortholog, is important for pancreatic islet cell development in zebrafish.
Mol. Genet. Metab.
PUBLISHED: 09-24-2010
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The adrenal cortex and pancreatic islets have endocrine functions, producing steroid-based hormones and insulin, respectively. Cells of the adrenal cortex originate in the mesoderm while the cells of pancreatic islets originate in the endoderm. The zebrafish is a powerful model for understanding organ development due to its ease of genetic and molecular manipulation, transparent embryos, and large number of progeny for statistically powerful experiments. Like humans, the zebrafish pancreas has both exocrine and endocrine functions; unlike humans, there is only one endocrine islet cell group, instead of multiple islets. Using an eGFP-transgenic line of zebrafish, we have observed that the steroidogenic factor 1 (SF1) ortholog, ff1b, which is critical for adrenal cortex development and function in the zebrafish, is also implicated in zebrafish pancreatic islet development. We show that interruption of ff1b expression using an ff1b-morpholino (MO) disrupts development of insulin expressing cells. We conclude that ff1b-MO alters pancreatic islet development in zebrafish, demonstrating the utility of the zebrafish as a model for studying pancreatic development. This work is consistent with previous studies in mouse and human that have suggested SF1 participates in the vascular and ductal development of the pancreas, and disruption of SF1 function leads to abnormal development of the pancreatic islets due to poor vascularization.
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The effects of hyperglycemia on adrenal cortex function and steroidogenesis in the zebrafish.
Mol. Genet. Metab.
PUBLISHED: 09-24-2010
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Since the 1950s, scientists have attempted to characterize the relationship between diabetes mellitus (DM) and the hypothalamic-pituitary-adrenal (HPA) axis. Similar complications are seen in patients with diabetes and Cushings syndrome, leading some to suggest that an underlying abnormality in the HPA axis may be responsible among those with DM. By inducing hyperglycemia in a zebrafish animal model, we show a direct correlation between glucose and cortisol levels.
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One-pot syntheses of chromeno[3,4-c]pyrrole-3,4-diones via Ugi-4CR and intramolecular Michael addition.
Org. Lett.
PUBLISHED: 09-23-2010
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One-pot and diastereoselective syntheses of diverse chromeno[3,4-c]pyrrole-3,4-diones from readily available starting materials were achieved via sequential Ugi-4CR and intramolecular Michael addition.
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Identification and expression analysis of mical family genes in zebrafish.
J Genet Genomics
PUBLISHED: 05-27-2010
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Mical (molecule interacting with CasL) represent a conserved family of cytosolic multidomain proteins that has been shown to be associated with a variety of cellular processes, including axon guidance, cell movement, cell-cell junction formation, vesicle trafficking and cancer cell metastasis. However, the expression and function of these genes during embryonic development have not been comprehensively characterized, especially in vertebrate species, although some limited in vivo studies have been carried out in neural and musculature systems of Drosophila and in neural systems of vertebrates. So far, no mical family homologs have been reported in zebrafish, an ideal vertebrate model for the study of developmental processes. Here we report eight homologs of mical family genes in zebrafish and their expression profiles during embryonic development. Consistent with the findings in Drosophila and mammals, most zebrafish mical family genes display expression in neural and musculature systems. In addition, five mical homologs are detected in heart, and one, micall2a, in blood vessels. Our data established an important basis for further functional studies of mical family genes in zebrafish, and suggest a possible role for mical genes in cardiovascular development.
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Genetic approach to evaluate specificity of small molecule drug candidates inhibiting PLK1 using zebrafish.
Mol Biosyst
PUBLISHED: 05-19-2010
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During the preclinical drug discovery process it remains a challenge to enable early elimination of candidate molecules that may have non-specific, off-target activities. Here, we use whole zebrafish embryo assays coupled with genetic analysis to address this issue. PLK1 (Polo-like kinase 1) is one of the key regulators that control mitotic entry, spindle assembly, chromosome segregation, and cytokinesis in the cell cycle. Since plk1 expression is abnormally up-regulated in several tumors, it is regarded as a good target for cancer therapy. A number of small-molecule inhibitors targeting PLK1 have been developed as reagents and anticancer drug candidates. It will be interesting to determine if these inhibitors indeed specifically target PLK1 in vivo. Bioinformatics analysis revealed that the zebrafish and human genomes share high homology across all PLK family members. In particular, PLK1 has a nearly identical 3-D structure between zebrafish and human. We selected three published PLK1 inhibitors, LFM-A13, ON01910, and thiazole-carboxamide 10A in our assay. When added at 2-cell stage, all of these inhibitors prevented embryos from dividing and caused cells to fuse into one large cell. When added at the later stage during zygotic mRNA transcription program initiation, embryos survived for 3 days but showed different phenotypes for each compound. Embryos treated with LFM-A13 appeared relatively normal. Embryos treated with ON01910 failed to properly develop trunk and tail regions while the head structure was unaffected. Embryos treated with thiazole-carboxamide 10A had a shorter body axis and deformed head structure. To determine which inhibitor is more selectively targeting PLK1, we inhibited PLK1 activity using anti-sense morpholino. Comparative analysis indicated that thiazole-carboxamide 10A could faithfully phenocopy zebrafish embryos genetically deficient of plk1. These findings demonstrate that these three PLK1 inhibitors, although well established by in vitro studies, have different off-target activities in vivo, and that thiazole-carboxamide 10A appears most specific to PLK1. Our studies suggest that zebrafish should be generally useful as an efficient in vivo model to evaluate specificity of small molecules designed to regulate any conserved target proteins through comparative analysis of genetic phenotypes.
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The effect of excess expression of GFP in a novel heart-specific green fluorescence zebrafish regulated by nppa enhancer at early embryonic development.
Mol. Biol. Rep.
PUBLISHED: 03-30-2010
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In order to study the impalpable effect of GFP in homozygous heart-specific GFP-positive zebrafish during the early stage, the researchers analyzed the heart function of morphology and physiology at the first 3 days after fertilization. This zebrafish line was produced by a large-scale Tol2 transposon mediated enhancer trap screen that generated a transgenic zebrafish with a heart-specific expression of green fluorescent protein (GFP)-tagged under control of the nppa enhancer. In situ hybridization experiments showed that the nppa:GFP line faithfully recapitulated both the spatial and temporal expressions of the endogenous nppa. Green fluorescence was intensively and specifically expressed in the myocardial cells located both in the heart chambers and in the atrioventricular canal. The embryonic heart of nppa:GFP line developed normally compared with those in the wild type. There was no difference between the nappa:GFP and wild type lines with respect to heart rate, overall size, ejection volume, and fractional shortening. Thus the excess expression of GFP in this transgenic line seemed to exert no detrimental effects on zebrafish hearts during the early stages.
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Scl isoforms act downstream of etsrp to specify angioblasts and definitive hematopoietic stem cells.
Blood
PUBLISHED: 02-25-2010
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Recent lineage studies suggest that hematopoietic stem cells (HSCs) may be derived from endothelial cells. However, the genetic hierarchy governing the emergence of HSCs remains elusive. We report here that zebrafish ets1-related protein (etsrp), which is essential for vascular endothelial development, also plays a critical role in the initiation of definitive hematopoiesis by controlling the expression of 2 stem cell leukemia (scl) isoforms (scl-alpha and scl-beta) in angioblasts. In etsrp morphants, which are deficient in endothelial and HSC development, scl-alpha alone partially rescues angioblast specification, arterial-venous differentiation, and the expression of HSC markers, runx1 and c-myb, whereas scl-beta requires angioblast rescue by fli1a to restore runx1 expression. Interestingly, when vascular endothelial growth factor (Vegf) signaling is inhibited, HSC marker expression can still be restored by scl-alpha in etsrp morphants, whereas the rescue of arterial ephrinb2a expression is blocked. Furthermore, both scl isoforms partially rescue runx1 but not ephrinb2a expression in embryos deficient in Vegf signaling. Our data suggest that downstream of etsrp, scl-alpha and fli1a specify the angioblasts, whereas scl-beta further initiates HSC specification from this angioblast population, and that Vegf signaling acts upstream of scl-beta during definitive hematopoiesis.
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TIF1gamma controls erythroid cell fate by regulating transcription elongation.
Cell
PUBLISHED: 02-23-2010
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Recent genome-wide studies have demonstrated that pausing of RNA polymerase II (Pol II) occurred on many vertebrate genes. By genetic studies in the zebrafish tif1gamma mutant moonshine we found that loss of function of Pol II-associated factors PAF or DSIF rescued erythroid gene transcription in tif1gamma-deficient animals. Biochemical analysis established physical interactions among TIF1gamma, the blood-specific SCL transcription complex, and the positive elongation factors p-TEFb and FACT. Chromatin immunoprecipitation assays in human CD34(+) cells supported a TIF1gamma-dependent recruitment of positive elongation factors to erythroid genes to promote transcription elongation by counteracting Pol II pausing. Our study establishes a mechanism for regulating tissue cell fate and differentiation through transcription elongation.
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In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.