Publications by authors named "Youdong Wang"

19 Publications

  • Page 1 of 1

Protocol development for discovery of angiogenesis inhibitors via automated methods using zebrafish.

PLoS One 2019 15;14(11):e0221796. Epub 2019 Nov 15.

Zebrafish Centre for Advanced Drug Discovery, Keenan Research Center, Li Ka Shing Knowledge Institute, St. Michael's Hospital, Unity Health Toronto, Toronto, Ontario, Canada.

Their optical clarity as larvae and embryos, small size, and high fecundity make zebrafish ideal for whole animal high throughput screening. A high-throughput drug discovery platform (HTP) has been built to perform fully automated screens of compound libraries with zebrafish embryos. A Tg(kdrl:EGFP) line, marking endothelial cell cytoplasm, was used in this work to help develop protocols and functional algorithms for the system, with the intent of screening for angiogenesis inhibitors. Indirubin 3' Monoxime (I3M), a known angiogenesis inhibitor, was used at various concentrations to validate the protocols. Consistent with previous studies, a dose dependant inhibitory effect of I3M on angiogenesis was confirmed. The methods and protocols developed here could significantly increase the throughput of drug screens, while limiting human errors. These methods are expected to facilitate the discovery of novel anti-angiogenesis compounds and can be adapted for many other applications in which samples have a good fluorescent signal.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0221796PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6857904PMC
March 2020

Bi-allelic GOT2 Mutations Cause a Treatable Malate-Aspartate Shuttle-Related Encephalopathy.

Am J Hum Genet 2019 09 15;105(3):534-548. Epub 2019 Aug 15.

On behalf of "United for Metabolic Diseases," 1105AZ Amsterdam, the Netherlands; Department of Laboratory Medicine, Translational Metabolic Laboratory, Radboud University Medical Centre, 6525 GA Nijmegen, the Netherlands. Electronic address:

Early-infantile encephalopathies with epilepsy are devastating conditions mandating an accurate diagnosis to guide proper management. Whole-exome sequencing was used to investigate the disease etiology in four children from independent families with intellectual disability and epilepsy, revealing bi-allelic GOT2 mutations. In-depth metabolic studies in individual 1 showed low plasma serine, hypercitrullinemia, hyperlactatemia, and hyperammonemia. The epilepsy was serine and pyridoxine responsive. Functional consequences of observed mutations were tested by measuring enzyme activity and by cell and animal models. Zebrafish and mouse models were used to validate brain developmental and functional defects and to test therapeutic strategies. GOT2 encodes the mitochondrial glutamate oxaloacetate transaminase. GOT2 enzyme activity was deficient in fibroblasts with bi-allelic mutations. GOT2, a member of the malate-aspartate shuttle, plays an essential role in the intracellular NAD(H) redox balance. De novo serine biosynthesis was impaired in fibroblasts with GOT2 mutations and GOT2-knockout HEK293 cells. Correcting the highly oxidized cytosolic NAD-redox state by pyruvate supplementation restored serine biosynthesis in GOT2-deficient cells. Knockdown of got2a in zebrafish resulted in a brain developmental defect associated with seizure-like electroencephalography spikes, which could be rescued by supplying pyridoxine in embryo water. Both pyridoxine and serine synergistically rescued embryonic developmental defects in zebrafish got2a morphants. The two treated individuals reacted favorably to their treatment. Our data provide a mechanistic basis for the biochemical abnormalities in GOT2 deficiency that may also hold for other MAS defects.
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http://dx.doi.org/10.1016/j.ajhg.2019.07.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6732527PMC
September 2019

Glutaminase Deficiency Caused by Short Tandem Repeat Expansion in .

N Engl J Med 2019 04;380(15):1433-1441

From Amsterdam University Medical Centers, University of Amsterdam, Departments of Clinical Chemistry, Pediatrics, and Clinical Genetics, Emma Children's Hospital, Amsterdam Gastroenterology and Metabolism (A.B.P.K., R.L., J.K., J. Meijer, L.A.T., M.T., M.W., R.J.A.W., H.R.W., C.D.M.K.), and United for Metabolic Diseases (A.B.P.K., R.J.A.W., H.R.W., C.D.M.K.), Amsterdam, and the Department of Neurology, Brain Center Rudolf Magnus, University Medical Center Utrecht (J.J.F.A.V., J.H.V.), and the Project MinE ALS Sequencing Consortium (J.J.F.A.V., J.H.V.), Utrecht - all in the Netherlands; the Departments of Biochemistry and Molecular Biology and Medical Genetics, Cumming School of Medicine, and Alberta Children's Hospital Research Institute, University of Calgary, Calgary (M.T.-G.), Centre for Molecular Medicine and Therapeutics, BC Children's Hospital Research Institute (P.A.R., M.J.J., M.S.K., J. MacIsaac, W.W.W., C.D.M.K.), the Faculty of Pharmaceutical Sciences (B.I.D., G.E.B.W., C.J.R.), and the Departments of Medical Genetics (C.M., I.-S.R.-B., W.W.W.) and Pediatrics (C.D.M.K.), University of British Columbia, Vancouver, the Zebrafish Centre for Advanced Drug Discovery, St. Michael's Hospital and University of Toronto (K.B.-A., F.K., M.L., Y.W., X.-Y.W.), the Centre for Applied Genomics, Genetics and Genome Biology, the Hospital for Sick Children (C.N., S.W.S., B.T., R.K.C.Y.), and the Department of Molecular Genetics (C.N., S.W.S., R.K.C.Y.), the McLaughlin Centre (S.W.S.), and the Departments of Medicine, Physiology, and Laboratory Medicine and Pathobiology, Institute of Medical Science (X.-Y.W.), University of Toronto, Toronto, and the Division of Medical Genetics, Department of Pediatrics, Children's Hospital Eastern Ontario, University of Ottawa, Ottawa (J.S.W., M.T.G.) - all in Canada; the Departments of Medicine and Physiology, National University of Singapore (M.A.P.), and the Translational Laboratory in Genetic Medicine, Agency for Science, Technology, and Research (M.A.P., B.S., X.X., J.Z.) - both in Singapore; Uppsala University, Department of Chemistry-Biomedical Center, Uppsala, Sweden (D.D.); Illumina, San Diego, CA (E.D., M.A.E.); Gene Structure and Disease Section, Laboratory of Cell and Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD (B.H., D.K., K.U.); and the Department of Clinical Inherited Metabolic Disorders, Birmingham Children's Hospital, Birmingham, United Kingdom (S.S.).

We report an inborn error of metabolism caused by an expansion of a GCA-repeat tract in the 5' untranslated region of the gene encoding glutaminase () that was identified through detailed clinical and biochemical phenotyping, combined with whole-genome sequencing. The expansion was observed in three unrelated patients who presented with an early-onset delay in overall development, progressive ataxia, and elevated levels of glutamine. In addition to ataxia, one patient also showed cerebellar atrophy. The expansion was associated with a relative deficiency of messenger RNA transcribed from the expanded allele, which probably resulted from repeat-mediated chromatin changes upstream of the repeat. Our discovery underscores the importance of careful examination of regions of the genome that are typically excluded from or poorly captured by exome sequencing.
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http://dx.doi.org/10.1056/NEJMoa1806627DOI Listing
April 2019

Simultaneous ultra-high frequency photoacoustic microscopy and photoacoustic radiometry of zebrafish larvae .

Photoacoustics 2018 Dec 1;12:14-21. Epub 2018 Sep 1.

Department of Physics, Ryerson University, Toronto, M5B 2K3, Canada.

With their optically transparent appearance, zebrafish larvae are readily imaged with optical-resolution photoacoustic (PA) microscopy (OR-PAM). Previous OR-PAM studies have mapped endogenous chromophores (e.g. melanin and hemoglobin) within larvae; however, anatomical features cannot be imaged with OR-PAM alone due to insufficient optical absorption. We have previously reported on the photoacoustic radiometry (PAR) technique, which can be used simultaneously with OR-PAM to generate images dependent upon the optical attenuation properties of a sample. Here we demonstrate application of the duplex PAR/PA technique for label-free imaging of the anatomy and vasculature of zebrafish larvae in vivo at 200 and 400 MHz ultrasound detection frequencies. We then use the technique to assess the effects of anti-angiogenic drugs on the development of the larval vasculature. Our results demonstrate the effectiveness of simultaneous PAR/PA for acquiring anatomical images of optically transparent samples in vivo, and its potential applications in assessing drug efficacy and embryonic development.
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http://dx.doi.org/10.1016/j.pacs.2018.08.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6139000PMC
December 2018

Development of a zebrafish sepsis model for high-throughput drug discovery.

Mol Med 2017 07 7;23:134-148. Epub 2017 Jun 7.

Zebrafish Centre for Advanced Drug Discovery, St. Michael's Hospital, 209 Victoria St, Toronto, Ontario, Canada M5B 1T8.

Sepsis is a leading cause of death worldwide. Current treatment modalities remain largely supportive. Intervention strategies focused on inhibiting specific mediators of the inflammatory host response have been largely unsuccessful, a consequence of an inadequate understanding of the complexity and heterogeneity of the innate immune response. Moreover, the conventional drug development pipeline is time consuming and expensive and the low success rates associated with cell-based screens underline the need for whole organism screening strategies, especially for complex pathological processes. Here, we established an LPS-induced zebrafish endotoxemia model, which exhibits the major hallmarks of human sepsis including, edema and tissue/organ damage, increased vascular permeability and vascular leakage accompanied by an altered expression of cellular junction proteins, increased cytokine expression, immune cell activation and ROS production, reduced circulation and increased platelet aggregation. We tested the suitability of the model for phenotype-based drug screening using three primary readouts: mortality, vascular leakage, and ROS production. Preliminary screening identified fasudil, a drug known to protect against vascular leakage in murine models, as a lead hit thereby validating the utility of our model for sepsis drug screens. This zebrafish sepsis model has the potential to rapidly analyze sepsis associated pathologies and cellular processes in the whole organism, as well as to screen and validate large numbers of compounds that can modify sepsis pathology in .
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http://dx.doi.org/10.2119/molmed.2016.00188DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5522968PMC
July 2017

A Model of Excitotoxic Brain Injury in Larval Zebrafish: Potential Application for High-Throughput Drug Evaluation to Treat Traumatic Brain Injury.

Zebrafish 2016 06 30;13(3):161-9. Epub 2016 Mar 30.

1 Institute of Medical Sciences, University of Toronto , Toronto, Ontario, Canada .

Traumatic brain injury (TBI) is a leading cause of death and morbidity with no effective therapeutic treatments for secondary injury. Preclinical drug evaluation in rodent models of TBI is a lengthy process. In this regard, the zebrafish has numerous advantages to address the technical and time-dependent obstacles associated with drug evaluation. We developed a reproducible brain injury using glutamate excitoxicity in zebrafish larvae, a known initiator of delayed cell death in TBI. Glutamate challenge resulted in dose-dependent lethality over an 84-h observation period. We report significant decrease in locomotion (p < 0.0001) and mean velocity (p < 0.001) with 10 μM glutamate application as measured through automated 96-well plate behavioral analysis. Application of the NMDA receptor antagonist MK-801 (400 nM) or the calpain inhibitor, MDL-28170 (20 μM), resulted in significant recovery of locomotor function. A secA5-YFP transgenic line was used to visualize the localization of cell death due to glutamate exposure in vivo using confocal fluorescence microscopy. Our results indicate that zebrafish larvae exhibit responses to excitotoxic injury and pharmacotherapeutic intervention with pathophysiological relevance to mammalian excitotoxic brain injury. This system has potential to be applied as a high-throughput drug screening model to quickly identify candidate lead compounds for further evaluation.
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http://dx.doi.org/10.1089/zeb.2015.1188DOI Listing
June 2016

Chromosome Condensation 1-Like (Chc1L) Is a Novel Tumor Suppressor Involved in Development of Histiocyte-Rich Neoplasms.

PLoS One 2015 20;10(8):e0135755. Epub 2015 Aug 20.

Keenan Research Centre for Biomedical Science, Li Ka Shing Knowledge Institute, St. Michael's Hospital, Toronto, Ontario, Canada; Department of Medicine & Institute of Medical Science, University of Toronto, Toronto, Ontario, Canada; Department of Physiology, University of Toronto, Toronto, Ontario, Canada.

Human chromosomal region 13q14 is a deletion hotspot in prostate cancer, multiple myeloma, and chronic lymphocytic leukemia. This region is believed to host multiple tumor suppressors. Chromosome Condensation 1-like (CHC1L) is located at 13q14, and found within the smallest common region of loss of heterozygosity in prostate cancer. Decreased expression of CHC1L is linked to pathogenesis and progression of both prostate cancer and multiple myeloma. However, there is no direct evidence for CHC1L's putative tumor suppressing role in current literature. Presently, we describe the generation and characterization of Chc1L knockout mice. Chc1L-/- mice do not develop cancer at a young age, but bone marrow and spleen cells from 8-12 week-old mice display an exaggerated proliferative response. By approximately two years of age, knockout and heterozygote mice have a markedly increased incidence of tumorigenesis compared to wild-type controls, with tumors occurring mainly in the spleen, mesenteric lymph nodes, liver and intestinal tract. Histopathological analysis found that most heterozygote and knockout mice succumb to either Histiocytic Sarcoma or Histiocyte-Associated Lymphoma. Our study suggests that Chc1L is involved in suppression of these two histiocyte-rich neoplasms in mice and supports clinical data suggesting that CHC1L loss of function is an important step in the pathogenesis of cancers containing 13q14 deletion.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0135755PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4546397PMC
May 2016

Knockdown of ZNF403 inhibits cell proliferation and induces G2/M arrest by modulating cell-cycle mediators.

Mol Cell Biochem 2012 Jun 19;365(1-2):211-22. Epub 2012 Feb 19.

Key Laboratory of Cancer Proteomics of Chinese Ministry of Health, Xiangya Hospital and Cancer Research Institute, Central South University, Changsha 410008, Hunan Province, China.

ZNF403, also known as GGNBP2 (gametogenetin binding protein 2), is a highly conserved gene implicated in spermatogenesis. However, the exact biological function of ZNF403 is not clear. In this study, we identified the role of ZNF403 in cell proliferation and cell-cycle regulation by utilizing short hairpin RNA (shRNA)-mediated knockdown. ZNF403-specific shRNA expressing helper-dependent adenoviral vector (HD-Ad-ZNF403-shRNA) was constructed and transduced human cell lines. ZNF403 mRNA and protein expression levels were inhibited as evidenced by real-time PCR and western blot analyses. Noticeably, we found that knockdown of ZNF403 expression suppressed cell proliferation compared to the non-target shRNA and vector controls. Furthermore, cell-cycle analysis demonstrated that downregulation of ZNF403 promoted G2/M cell-cycle arrest in a dose-dependent manner. Moreover, human cell-cycle real-time PCR array revealed that ZNF403 knockdown influenced the expression profile of genes in cell-cycle regulation. Among these genes, western blot analysis confirmed the protein up-regulation of p21 and down-regulation of MCM2 in response to the ZNF403 knockdown. Additionally, knockdown of ZNF403 also showed an anti-carcinogenetic effect on anchorage-independent growth by colony formation assay and tumor cell migration by wound-healing assay with human laryngeal cancer cell line Hep-2 cells. Altogether, our findings suggest an essential role of ZNF403 in cell proliferation and provide a new insight into the function of ZNF403 in regulating the G2/M cell-cycle transition.
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http://dx.doi.org/10.1007/s11010-012-1262-6DOI Listing
June 2012

Expression and characterization of recombinant Rhizopus oryzae lipase for enzymatic biodiesel production.

Bioresour Technol 2011 Oct 24;102(20):9810-3. Epub 2011 Jul 24.

College of Chemical Engineering, Nanjing Forestry University, Nanjing 210037, China.

The Rhizopus oryzae lipase containing prosequence was expressed in Pichia pastoris. Recombinant lipase subunit showed a molecular mass of 32 kDa. The maximum activity of recombinant lipase obtained from Mut(s) recombinant was 90 IU/ml. The enzyme was stable in broad ranges of temperatures and pH, with the optimal temperature at 35 °C and pH 7.0. The crude recombinant R. oryzae lipase can be directly used for the transesterification of plant oils at high-water content of 60-100% (w/w) based on oil weight. The addition of 80% water to the transesterification systems resulted in the yield of methyl ester of 95%, 94% and 92% after 72 h using soybean oil, Jatropha curcas seed raw oil and Pistacia chinensis seed raw oil as raw material, respectively. These results indicate that the recombinant lipase is an effective biocatalyst for enzymatic biodiesel production.
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http://dx.doi.org/10.1016/j.biortech.2011.07.070DOI Listing
October 2011

Rosuvastatin, identified from a zebrafish chemical genetic screen for antiangiogenic compounds, suppresses the growth of prostate cancer.

Eur Urol 2010 Sep 22;58(3):418-26. Epub 2010 May 22.

Keenan Research Center, Li Ka Shing Knowledge Institute, St. Michael's Hospital and Department of Medicine, University of Toronto, Toronto, Ontario, Canada.

Background: Prostate cancer (PCa) is the most common malignancy in males in Western countries. Despite improvements in standard treatments such as surgery, radiotherapy, and chemotherapy, many patients still progress to advanced stages. Recent clinical trials have shown encouraging results regarding the application of angiogenic inhibitors in the treatment of angiogenesis-dependent diseases, paving the way for novel PCa therapies.

Objective: To identify new antiangiogenic compounds and examine their therapeutic potential in models of PCa.

Design, Setting, And Participants: We performed a chemical genetic screen in developing zebrafish embryos to identify small molecules inhibiting zebrafish angiogenesis. Transgenic Tg(flk1:EGFP) zebrafish embryos were used in the screening of the Spectrum Collection compound library. Subsequently, the antiangiogenic mechanism of an identified lead compound, rosuvastatin, was studied by conducting endothelial cell function assays and examining antitumor efficacy in a PCa xenograft mouse model. MEASUREMENTS, RESULTS AND LIMITATIONS: Seven lead compounds, including isorotenone, dihydromunduletone, aristolochic acid, simvastatin, mevastatin, lovastatin, and rosuvastatin, were identified to inhibit the growth of the zebrafish intersegmental vessels. Of these seven leads, rosuvastatin was further evaluated for its antiangiogenic mechanism and anticancer efficacy. Rosuvastatin decreased the viability of the human umbilical endothelial cells (HUVECs) (one-half inhibitory concentration: 5.87 microM) by inducing G(1) phase arrest and promoting apoptosis. Moreover, rosuvastatin remarkably inhibited the migration of HUVECs and dose-dependently inhibited the HUVEC capillary-like tube formation in vitro. Furthermore, we demonstrated that rosuvastatin suppressed xenografted PPC-1 prostate tumors in nonobese diabetic severe combined immunodeficiency (NOD-SCID) mice associated with decreased microvessel density (MVD) and tumor cell apoptosis.

Conclusions: Collectively, our data suggest that rosuvastatin possesses antiangiogenic and antitumor activities and has therapeutic potential for the treatment of PCa. This study represents the first zebrafish antiangiogenic chemical genetic screen to identify a lead compound that targets cancer angiogenesis.
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http://dx.doi.org/10.1016/j.eururo.2010.05.024DOI Listing
September 2010

Characterisation and biological activities of proanthocyanidins from the barks of Pinus massonian and Acacia mearnsii.

Nat Prod Res 2010 Apr;24(6):590-8

College of Chemical Engineering, Nanjing Forestry University, Nanjing, China.

The biological activities and characterisation of proanthocyanidins (PAs) from the barks of Pinus massonian and Acacia mearnsii have been studied in our research. The free-radical scavenging activity of the PAs was measured by means of the DPPH method, and it was clear that the PA product had a strong radical scavenging ability. Furthermore, anti-tumour activities of PAs on different human cancer cells were investigated. The results indicated that PAs from the bark of A. mearnsii had better anti-tumour activities than those from the bark of P. massonian, and the PAs extracted from the ethyl acetate fraction had better anti-tumour activities than those from the water fraction. PAs from the bark of A. mearnsii in an ethyl acetate fraction (PAE) had an effective inhibition on human mammary cancer cells (MDA- MB-231) and human liver cancer cells (BEL-7402), a weak effect on human cervical cancer cells (Hela), but no effect on human lung cancer cells (A549); while the PAs from the bark of A. mearnsii in a water fraction (PAW) had weak effect on MDA- MB-231, Hela and A549, but no effect on BEL-7402. PAs from the bark of P. massonian in ethyl acetate fraction (PPE) had weak effect on Hela and BEL-7402; whereas the PAs from the bark of P. massonian in a water fraction (PPW) had a weak effect on Hela, but no effect on the other cells. PAs were characterised by HPLC- ESI-MS analysis and the PA dimers and trimers were identified, respectively.
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http://dx.doi.org/10.1080/14786410903194472DOI Listing
April 2010

Enhanced adaptive immunity in mice lacking the immunoinhibitory adaptor Hacs1.

FASEB J 2010 Mar 18;24(3):947-56. Epub 2009 Nov 18.

St. Michael's Hospital, 30 Bond St., Queen Wing, Rm 8-019, Toronto, ON, Canada M5B 1W8.

Hacs1, a SH3 and SAM domain-containing adaptor protein, is up-regulated by IL-4 in activated B cells and strongly expressed in dendritic cells. To elucidate the function of Hacs1 in immune regulation, we generated Hacs1(-/-) mice by deletion of the SH3 and SAM domains. Hacs1(-/-) mice were viable and fertile and had normal bone marrow B-cell development and normal splenic T- and B-cell populations. However, adult Hacs1(-/-) mice had increased peritoneal B1a cells (IgM(+)CD5(+)). On immunization with T-cell-independent antigen TNP-Ficoll, Hacs1(-/-) mice had increased production of anti-TNP IgM and IgG3. Purified splenic B cells from Hacs1(-/-) mice showed increased cell proliferation on BCR (B-cell receptor) stimulation. We further demonstrate that the Hacs1(-/-) B cells had increased global tyrosine phosphorylation, including tyrosine kinases Lyn and Akt. Both T-helper type 1 (T(h)1) and T-helper type 2 (T(h)2) humoral responses were enhanced in Hacs1(-/-) mice. In vitro bone marrow-derived Hacs1(-/-) dendritic cells showed increased IL-12 production on stimulation with ovalbumin (OVA). This study suggests that Hacs1 is an immunoinhibitory adaptor that might be a useful target for immune suppression therapy.-Wang, D., Stewart, A. K., Zhuang, L., Zhu, Y., Wang, Y., Shi, C., Keating, A., Slutsky, A., Zhang, H., Wen, X.-Y. Enhanced adaptive immunity in mice lacking the immunoinhibitory adaptor Hacs1.
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http://dx.doi.org/10.1096/fj.09-140806DOI Listing
March 2010

Effect of digital problem-based learning cases on student learning outcomes in ophthalmology courses.

Arch Ophthalmol 2009 Sep;127(9):1211-4

Department of Ophthalmology, The Fourth Affiliated Hospital of China Medical University, Heping District, Shenyang City 110005, China.

Objective: To assess the impact of digital problem-based learning (PBL) cases on student learning in ophthalmology courses.

Methods: Ninety students were randomly divided into 3 classes (30 students per class). The first class studied under a didactic model. The other 2 classes were divided into 6 groups (10 students per group) and received PBL teaching; 3 groups studied via cases presented in digital form and the others studied via paper-form cases. The results of theoretical and case analysis examinations were analyzed using the chi(2) test. Student performance on the interval practice was analyzed using the Kruskal-Wallis test. Questionnaires were used to evaluate student and facilitator perceptions.

Results: Students in the digital groups exhibited better performance in the practice procedures according to tutorial evaluations compared with the other groups (P < .05). The 2 PBL classes had significantly higher mean results of theoretical and case analysis examinations (P < .001), but there was no significant difference between the 2 PBL classes. Ninety-three percent of students in the digital groups (vs 73% in the paper groups) noted that the cases greatly stimulated their interest.

Conclusions: Introducing PBL into ophthalmology could improve educational quality and effectiveness. Digital PBL cases stimulate interest and motivate students to further improve diagnosis and problem-handling skills.
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http://dx.doi.org/10.1001/archophthalmol.2009.110DOI Listing
September 2009

Comparative analyses of genomic imprinting and CpG island-methylation in mouse Murr1 and human MURR1 loci revealed a putative imprinting control region in mice.

Gene 2006 Jan 21;366(1):77-86. Epub 2005 Nov 21.

Department of Biomolecular Sciences, Faculty of Medicine, Saga University, Saga 849-8501, Japan.

Human MURR1 is an orthologue of mouse Murr1 gene, which was previously reported to be imprinted only in adult brain with a maternal allele-predominant expression and to contain another imprinted gene, U2af1-rs1, in the first intron. Human MURR1 was found not to harbor the U2af1-rs1 orthologue and to be expressed biallelically in tissues, including adult brain. Three genes identified around Murr1 and their orthologues around MURR1 were expressed biallelically. These findings suggest that the mouse imprinting locus is limited to a small region and the introduction of U2af1-rs1 in mouse causes the imprinting of this locus. The CpG island (CGI) at U2af1-rs1 with maternal methylation was the only differentially methylated region among CGIs found in these loci. Detailed methylation analyses of the U2af1-rs1 CGI in germ cells led to identification of a region with oocyte-specific methylation. These results suggest that this region is the imprinting control region of the Murr1/U2af1-rs1 locus in mouse.
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http://dx.doi.org/10.1016/j.gene.2005.08.020DOI Listing
January 2006

The mouse Murr1 gene is imprinted in the adult brain, presumably due to transcriptional interference by the antisense-oriented U2af1-rs1 gene.

Mol Cell Biol 2004 Jan;24(1):270-9

Department of Biomolecular Sciences, Saga Medical School, Saga 849-8501, Japan.

The mouse Murr1 gene contains an imprinted gene, U2af1-rs1, in its first intron. U2af1-rs1 shows paternal allele-specific expression and is transcribed in the direction opposite to that of the Murr1 gene. In contrast to a previous report of biallelic expression of Murr1 in neonatal mice, we have found that the maternal allele is expressed predominantly in the adult brain and also preferentially in other adult tissues. This maternal-predominant expression is not observed in embryonic and neonatal brains. In situ hybridization experiments that used the adult brain indicated that Murr1 gene was maternally expressed in neuronal cells in all regions of the brain. We analyzed the developmental change in the expression levels of both Murr1 and U2af1-rs1 in the brain and liver, and we propose that the maternal-predominant expression of Murr1 results from transcriptional interference of the gene by U2af1-rs1 through the Murr1 promoter region.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC303337PMC
http://dx.doi.org/10.1128/mcb.24.1.270-279.2004DOI Listing
January 2004

Characterization and imprinting status of OBPH1/Obph1 gene: implications for an extended imprinting domain in human and mouse.

Genomics 2002 Dec;80(6):575-84

Department of Biomolecular Sciences, Saga Medical School, 5-1-1 Nabeshima, Saga, 849-8501, Japan.

Human 11p15.5, as well as its orthologous mouse 7F4/F5, is known as the imprinting domain extending from IPL/Ipl to H19. OBPH1 and Obph1 are located beyond the presumed imprinting boundary on the IPL/Ipl side. We determined full-length cDNAs and complete genomic structures of both orthologues. We also investigated their precise imprinting and methylation status. The orthologues resembled each other in genomic structure and in the position of the 5' CpG island and were expressed ubiquitously. OBPH1 and Obph1 were predominantly expressed from the maternal allele only in placenta, with hypo- and not differentially methylated 5' CpG islands in both species. These results suggested that the imprinting domain would extend beyond the presumed imprinting boundary and that methylation of the 5' CpG island was not associated with the imprinting status in either species. It remains to be elucidated whether the gene is under the control of the KIP2/LIT1 subdomain or is regulated by a specific mechanism. Analysis of the precise genomic sequence around the region should help resolve this question.
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http://dx.doi.org/10.1006/geno.2002.7006DOI Listing
December 2002

Domain regulation of imprinting cluster in Kip2/Lit1 subdomain on mouse chromosome 7F4/F5: large-scale DNA methylation analysis reveals that DMR-Lit1 is a putative imprinting control region.

Genome Res 2002 Dec;12(12):1860-70

Department of Biochemistry, Saga Medical School, Saga, Saga 849-8501, Japan.

Mouse chromosome 7F4/F5, where the imprinting domain is located, is syntenic to human 11p15.5, the locus for Beckwith-Wiedemann syndrome. The domain is thought to consist of the two subdomains Kip2 (p57(kip2))/Lit1 and Igf2/H19. Because DNA methylation is believed to be a key factor in genomic imprinting, we performed large-scale DNA methylation analysis to identify the cis-element crucial for the regulation of the Kip2/Lit1 subdomain. Ten CpG islands (CGIs) were found, and these were located at the promoter sites, upstream of genes, and within intergenic regions. Bisulphite sequencing revealed that CGIs 4, 5, 8, and 10 were differentially methylated regions (DMRs). CGIs 4, 5, and 10 were methylated paternally in somatic tissues but not in germ cells. CGI8 was methylated in oocyte and maternally in somatic tissues during development. Parental-specific DNase I hypersensitive sites (HSSs) were found near CGI8. These data indicate that CGI8, called DMR-Lit1, is not only the region for gametic methylation but might also be the imprinting control region (ICR) of the subdomain.
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http://dx.doi.org/10.1101/gr.110702DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC187562PMC
December 2002

Identification of a novel isoform of Murr1 transcript, U2mu, which is transcribed from the portions of two closely located but oppositely oriented genes.

Genes Genet Syst 2002 Oct;77(5):377-81

Department of Biomolecular Sciences, Saga Medical School, Japan.

Here we identified a novel transcript in mouse that is transcribed from the portions of two independent genes, U2af1-rs1 and Murr1, and we designated it U2mu. The U2af1-rs1 gene is located in the intron and transcribed in the opposite direction from the Murr1 gene on the proximal region of mouse chromosome 11. The U2mu cDNA sequence is derived from three genomic regions--an intron of the Murr1 gene, an antisense sequence of U2af1-rs1 gene, and the last exon of Murr1 gene--in the order of 5' to 3'. The U2mu transcript of 2.8 kb is expressed ubiquitously in adult mice. It is transcribed biallelically, and is not imprinted, in neonatal and adult mice.
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http://dx.doi.org/10.1266/ggs.77.377DOI Listing
October 2002