Dept of Biochemistry

DEPT OF BIOCHEMISTRY

Researcher : Agrawal KR

List of Research Outputs

Yang S., Agrawal K.R., Lam T.W., Sham P.C., Cheah K.S.E. and Wang J.J., Novel Profile Hidden Markov Model to Predict MicroRNAs and their Targets Simutaneously, 14th Research Postgraduate Symposium, The University of Hong Kong, December 2-3. 2009.

Yang S., Agrawal K.R., Lam T.W., Sham P.C., Cheah K.S.E. and Wang J.J., Novel Profile Hidden Markov Model to predict microRNAs in Degenerative Disc Disease. University Grants Council site visit for Area of Excellence Programme "Developmental Genomics & Skeletal Research". January 25, 2010.

Researcher : Au YK

List of Research Outputs

Cheah K.S.E., Au Y.K., Szeto Y.Y., Wynn S., Chan Y.S., Cheung K.M.C., Chan W.Y., Lovell-Badge R.H., Chan D. and Fritzsch B., Molecular and developmental insights into the pathogenesis of the SOX9Y440Xcampomelic dysplasia mutation, 16th International Society of Developmental Biologists Congress 6-10th September 2009 Edinburgh International Conference Centre, UK . 2009.

Researcher : Cai S

List of Research Outputs

Cai S., Pan Y., Fu X.B., Lei Y.H. and Sheng Z.Y., Dedifferentiation of human epidermal keratinocytes induced by UV in vitro, Journal of health science. Japan, Pharmaceutical Society of Japan, 2009, 55: 709-719.

Cai S., Pan Y., Fu X.B., Sun T.Z., Lei Y.H. and Sheng Z.Y., Dedifferentiation of human epidermal keratinocytes induced by combined heat and basic fibroblast growth factor treatment, 熱損傷與bFGF聯合處理誘導表皮細胞的去分化過程, Infection,Inflammation and Repair. 感染炎症修復, Beijing, 2010, 11: 4-7.

Cai S., Shum D.K.Y., Chan Y.S. and Fu X.B., Outstanding Abstract Prize (Poster Category), Research Center of Heart, Brain, Hormone & Healthy Aging. 2010.

Cai S., State Science & Technology Awards, 國家科學技術進步二等獎, China's State Council. 中華人民共和國國務院, 2009.

Researcher : Cham WC

List of Research Outputs

Cham W.C., Ma C.W., Zhang Y., Xie H., Yung W.H., Chan Y.S. and Shum D.K.Y., The Regulatory Role of Heparanase in Synaptic Plasiticity at Hippocampus, 14th Research Postgraduate Symposium, December 2 & 3, 2009, The University of Hong Kong. 2009.

Cham W.C., Ma C.W., Zhang Y., Xie H., Yung W.H., Chan Y.S. and Shum D.K.Y., The regulatory role of heparanase of synaptic plasticity in hippocampus, Society Neuroscience Abstract (USA). 2009, 318.3/C4.

Ma C.W., Zhang Y., Cham W.C., Chan Y.S. and Shum D.K.Y., Role of heparanase in synaptic plasticity at the hippocampus and vestibular nucleus , J. Physiological Sciences (Suppl 1). [36^th Int’l Congress of International Union of Physiological Sciences Kyoto, Japan. July 27 - Aug. 1, 2009]. 2009, 59: 145.

Researcher : Cham WC

List of Research Outputs

Researcher : Chan C

List of Research Outputs

Shum K.T., Chan C., Leung C.M. and Tanner J.A., Aptamer-based Inhibitor of Sclerostin for Osteoporosis Therapy, 14th Research Postgraduate Symposium, December 2 & 3, 2009, The University of Hong Kong. 2009.

Researcher : Chan CH

List of Research Outputs

Chan C.H., Leung V.O.Y., Lam C.L.D., Mak J.C.W., Freeman C., Ip M.S.M. and Shum D.K.Y., Sulfated maltoheptaose reduces neutrophilic airway inflammation in a smoking rat model of chronic obstructive pulmonary disease , Fifth International Symposium on Healthy Aging: “Is Aging a Disease?” 6-7 March 2010 . 2010.

Ip C.H., Chan C.H. and Shum D.K.Y., Serglycin in the HL-60 degranulation product retains its sulfated chondroitin/dermatan moieties and affinity for neutrophil elastase, 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium, CUHK, Hong Kong, 15 May,. 2010.

Shum D.K.Y., Ip M.S.M., Chan C.H. and Leung O.Y.V., Compositions and Methods for Treating chronic Respiratory Inflammation. U.S. Provisional Application No. 61/308,597 filed on Feb 26, 2010 with the U.WS. Patent and Trademark Office. , 2010.

Researcher : Chan CP

List of Research Outputs

Chan C.P., Mak T.Y., Chin K.T. and Ng I.O.L., N-linked glycosylation is required for optimal proteolytic activation of membrance-bound transcription factor CREB-H, 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium, CUHK, Hong Kong, 15 May. 2010.

Chan C.P., Mak T.Y., Chin K.T., Ng I.O.L. and Jin D., N-linked glycosylation is required for optimal proteolytic activation of membrane-bound transcription factor CREB-H., J. Cell Sci.. The Company of Biologists, 2010, 123: 1438-1448.

Chan C.P., Mak T.Y., Chin K.T. and Jin D., Requirement of N-linked Glycosylation in the Luminal Domain for Optimal Proteolytic Activation of Liver-enriched transcription Factor CREB-H, 14th Research Postgraduate Symposium, December 2 & 3, 2009, The University of Hong Kong. 2009.

Chan C.P., Requirement of N-linked glycosylation for optimal proteolytic activation of liver-enriched transcription factor CREB-H, PhD Thesis , 2010.

Mak T.Y., Chan C.P. and Jin D., Differential stability and transcriptional activity of CREB3 subfamily transcription factors, 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium, CUHK, Hong Kong, 15 May,. 2010.

Siu K.L., Chan C.P., Chan C.S., Zheng B. and Jin D., Severe acute respiratory syndrome coronavirus nucleocapsid protein does not modulate transcription of human FGL2 gene., J. Gen. Virol.. 2009, 90: 2107-2113.

Researcher : Chan CP

List of Research Outputs

Chan C.P., Requirement of N-linked glycosylation for optimal proteolytic activation of liver-enriched transcription factor CREB-H, PhD Thesis , 2010.

Researcher : Chan CP

List of Research Outputs

Chan C.P., Requirement of N-linked glycosylation for optimal proteolytic activation of liver-enriched transcription factor CREB-H, PhD Thesis , 2010.

Researcher : Chan CSL

List of Research Outputs

Shum K.T., Chan C.S.L., Leung C.M. and Tanner J.A., Aptamer-mediated inhibition of sclerostin for osteoporosis therapy. , 5th Annual Meeting of the Oligonucleotide Therapeutics Society, Fukuoka, Japan . 2009.

Shum K.T., Chan C.S.L., Leung C.M. and Tanner J.A., G-Quadruplex Aptamers Inhibit Sclerostin for Osteoporosis Therapy, CUHK Croucher Advanced Study Institute “Structure-Based Screening and Design of Ligands for Protein Targets” . 2009.

Tanner J.A., Shum K.T. and Chan C.S.L., High-affinity nucleic acid aptamers against sclerostin protein, US Provisional Patent. USA, 2010, 61/349,058: 61/349,058.

Researcher : Chan CWW

List of Research Outputs

Chan C.W.W., ER Stress in the Pathogenesis of Osteochondrodysplasia, PhD Thesis. 2009.

Researcher : Chan CY

List of Research Outputs

Chan C.Y., Tsang S.W. and Yao K.M., Secreted PDZ Domain-containing Protein 2 (sPDZD2) Exerts Insulinotropic Effects on INS-1E Cells via a Protein Kinase A-Dependent Mechanism, Japan Society of Developmental Biology 2010 at Kyoto, Japan. 2010.

Chan C.Y., Secreted PDZ domain-containing protein 2 (sPDZD2) exerts insulinotropic effects on INS-1E cells via a protein kinase A - dependent mechanism, MPhil Thesis. 2009.

Researcher : Chan CYC

List of Research Outputs

Chan C.Y.C., Understanding Sedlin and the Molecular Basis of Spondyloepiphyseal Dysplasia Tarda, PhD Thesis. 2009.

Researcher : Chan D

Project Title:	Genome-wide identification and functional studies of genetic risk factors for low back pain and intervertebral disc degeneration
Investigator(s):	Chan D
Department:	Biochemistry
Source(s) of Funding:	AOSpine International - General Award
Start Date:	08/2007

Abstract:

To identify novel genetic risk factors for DDD, by performing a genome-wide association study on a Southern Chinese cohort; to perform functional studies of genetic risk factors using mouse as a model system.

Project Title:	Role of Indian hedgehog signaling in phalangeal joint formation and Brachydactylies
Investigator(s):	Chan D, Cheah KSE, Cheung MCH
Department:	Biochemistry
Source(s) of Funding:	General Research Fund (GRF)
Start Date:	01/2009

Abstract:

(1) To gain further insights into the role of IHH signaling in distal digit formation required for cellular differentiation and maintenance; (2) To understand the molecular mechanisms underlying the abnormal signaling capacity and range from the E95K mutation.

List of Research Outputs

Aladin K.D.M., Cheung K.M.C., Ngan A.H.W., Chan D., Leung Y.L., Lim C.T., Luk K.D.K. and Lu W.W., Nanostructure of Collagen Fibrils in Human Nucleus Pulposus and Its Correlation with Macroscale Tissue Mechanics, J. Ortho. Res.. 2009, 28: 497-502.

Aladin Kaderbatcha D.M., Cheung K.M.C., Ngan A.H.W., Chan D., Leung Y.L., Lim C.T., Luk K.D.K. and Lu W.W., Nano-structure of collagen fibrils in human nucleus pulposus and its correlation with macroscale tissue mechanics, Journal of Orthopedic Research. 2010, 28: 497-502.

Chan C.W., Gantenbein-Ritter B., Leung Y.L., Chan D., Cheung K.M.C. and Ito K., Cryopreserved intervertebral disc with injected bone marrow-derived stromal cells: a feasibility study using organ culture, The Spine Journal. 2010, 10: 486-496.

Chan C.W., Gantenbein-Ritter B., Leung Y.L., Chan D., Cheung K.M.C. and Ito K., Cryopreserved intervertebral disc with injected bone marrow–derived stromal cells: a feasibility study using organ culture, The Spine Journal. 2010, 10: 486–496.

Chan C.W., Lam S.K.L., Leung Y.L., Chan D., Luk K.D.K. and Cheung K.M.C., Minimizing cryopreservation-induced loss of disc cell activity for storage of whole intervertebral discs, European Cells and Materials . 2010, 19: 273-283.

Chan D., Bone Pathology, JAMA. 2010, 303: 1098-1099.

Cheah K.S.E., Wang C., Tan Z., Melhado I.G., Niu B., Tsang K.Y., Zhang M...Q..., Ron D... and Chan D., Alleviating ER stress in chondrocytes: survival strategies with double-edged developmental consequences , 43rd Annual Meeting for the Japanese Society of Developmental Biologists Jointly Sponsored by the Asia-Pacific Developmental Biology Network, Kyoto, Japan, 20 June 2010.

Cheah K.S.E., Wang C., Tan Z., Melhado I.G., Niu B., Tsang K.Y., Zhang M...Q..., Ron D. and Chan D., Genetic and genomic analyses of chondrocyte adaptation to ER stress, The Genetics Society Spring Meeting “Mouse Genetics – Think Globally, Act Locally” The Wellcome Trust Conference Centre, Hinxton, UK, 23 April 2010 . 2010.

Cheah K.S.E., Au Y.K., Szeto Y.Y., Wynn S., Chan Y.S., Cheung K.M.C., Chan W.Y., Lovell-Badge R.H., Chan D. and Fritzsch B., Molecular and developmental insights into the pathogenesis of the SOX9Y440Xcampomelic dysplasia mutation, 16th International Society of Developmental Biologists Congress 6-10th September 2009 Edinburgh International Conference Centre, UK . 2009.

Cheng H.W., Tsui Y.K., Cheung K.M.C., Chan D. and Chan B.P., Decellularization of chondrocyte-encapsulated collagen microspheres - a 3D model to study the effects of acellular matrix on stem cell fate, Tissue Engineering Part C Methods . 2009, 15(4): 697-706.

Cheng H.W., Tsui Y.K., Cheung K.M.C., Chan D. and Chan B.P., Decellularization of chondrocyte-encapsulated collagen microspheres. , WACBE (World Association for Chinese Biomedical Engineers) World Congress on Bioengineering 2009. 26-29 Jul 2009, Hong Kong, China. . 2009, 58.

Cheng H.W., Tsui Y.K., Cheung K.M., Chan D. and Chan B.P., Decellularization of chondrocyte-encapsulated collagen microspheres: a three-dimensional model to study the effects of acellular matrix on stem cell fate , Tissue Engineering, Part C Methods. 2009, 15(4): 697-706.

Cheng H.W., Chan D. and Chan B.P., Fabrication of an osteochondral construct with an intact interface using rabbit mesenchymal stem cells (rMSCs) and collagen gel, ISSCR 8th Annual Meeting. Jun 16-19, 2010, Moscone West, San Francisco, CA USA. . 2010, #Y-40, p239.

Choi M.Y., Chan C.C.Y., Chan D., Luk K.D.K., Cheah K.S.E. and Tanner J.A., Biochemical consequences of sedlin mutations that cause spondyloepiphyseal dysplasia tarda, Biochemical Journal. Great Britain, Biochemical Society, 2009, 423: 233-242.

Gao Y., Leung W.L., Wong S.Y.Y., Tam P.P.L., Chan D. and Cheah K.S.E., Type IIA Procollagen: A Positive Regulator of Nodal Signaling During Early Vertebrate Embryogenesis, 14th Research Postgraduate Symposium, December 2 & 3, 2009, The University of Hong Kong. 2009.

Gao Y., Leung W.L., Wong Y.Y., Chan D., Tam P.P.L. and Cheah K.S.E., Type IIA procollagen: A positive regulator of nodal signaling during embryogenesis, 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium, CUHK, Hong Kong, 15 May. 2010.

Garcia-Barcelo M.M., Yeung M.Y., Miao X.P., Tang S.M., Chen G., So M.T., Ngan E.S.W., Lui V.C.H., Chen Y., Liu X., Hui K.J.W.S., Li L., Guo W.H., Sun X.B., Tou J.F., Chan K.W., Wu X.Z., Song Y., Chan D., Cheung K.M.C., Chung P.H.Y., Wong K.K.Y., Sham P.C., Cherny S.S. and Tam P.K.H., Genome-wide association study identifies a susceptibility locus for biliary atresia on 10q24.2, Human Molecular Genetics. 2010, 19 (14): 2917-2925.

Guo S., Zhou J., Gao B., Hu J., Wang H., Meng J., Zhao X., Ma G., Lin C., Xiao Y., Tang W., Zhu X., Cheah K.S.E., Feng G., Chan D. and He L., Missense mutations in IHH impair Indian Hedgehog signaling in C3H10T1/2 cells: Implications for brachydactyly type A1, and new targets for Hedgehog signaling, Cell Mol Biol Lett. 2009, 15(1): 153-76.

Kao Y.P.P., Chan D., Cheah K.S.E., Cheung K.M.C., Ho D.W.H., Karppinen J., Leong J.C.Y., Yip S.P., Song Y. and Sham P.C., Genome-wide Association Study of Degenerative Disc Disease (DDD), 14th Research Postgraduate Symposium, December 2 & 3, 2009, The University of Hong Kong. 2009.

Lee K.S., Lam T.K., Song Y., Cheah K.S.E., Cheung K.M.C. and Chan D., In vivo study of Asporin, a genetic risk factor for Osteoarthritis and degenerative disc disease, 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium, CUHK, Hong Kong, 15 May,. 2010.

Leung Y.L., Tsui Y.K., Kao R.Y.T., Masuda K., Chan D. and Cheung K.M.C., Identifying therapeutic chemical agents for intervertebral disc degeneration by high throughput screening, 55th Annual Meeting of the Orthopaedic Research Society, New Orleans, USA. 2010.

Leung Y.L., Gao B., Melhado I.G., Leung K.K.H., Wynn S.L., Chan D. and Cheah K.S.E., Sox9 mediates gene expression specificity in growth plate chondrocytes via concomitant positive and negative regulation, 55th Annual Meeting of the Orthopaedic Research Society, New Orleans, USA. 2010.

Li Y.Y., Cheng H.W., Wong M.Y., Cheung K.M.C., Chan D. and Chan B.P., Collagen-mesenchymal stem cell microspheres for cartilage tissue engineering. , TERMIS 2nd World Congress, Seoul, Korea, Aug 31- 3 Sep 2009. . 2009, S69:222.

Li Y.Y., Cheng H.W., Chan P.M., Wong M.Y., Teng W.K., Chow S.T., Cheung K.M.C., Chan D. and Chan B.P., Repair of osteochondral defects with collagen-mesenchymal stem cell microspheres in a rabbit model. , ISSCR 8th Annual Meeting. Jun 16-19, 2010, Moscone West, San Francisco, CA USA. . 2010, #W-50, p250.

Liao H., Huang S., Cheung K.M.C., Chan D., Luk K.D.K. and Zhou G., Functional characterization of miRNA125b, miRNA140 and miRNA199a in chondrogenetic differentiation of mesenchymal stem cells, Cell Adhesion & Communication.. 2009, In press.

Lie Y.S., Chan W.C.W., Tsang K.Y., Cheah K.S.E. and Chan D., Impact of intracellular clearance of unfolded proteins on chondrocytes cell fate, 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium, CUHK, Hong Kong, 15 May,. 2010.

Lu L., Chan D., Cheah K.S.E. and Cheung M.C.H., Epigenetic Reprogramming of Fibroblasts into Osteo-chondroprogenitors, 14th Research Postgraduate Symposium, December 2 & 3, 2009, The University of Hong Kong. 2009.

Lu L., Cheah K.S.E., Chan D. and Cheung M.C.H., Epigenetic reprogramming of fibroblasts into osteo-chondroprogenitors, 43rd Annual Meeting for the Japanese Society of Developmental Biologists Kyoto, Japan, 20 June 2010 . 2010.

Lu W.W., Aladin Kaderbatcha D.M., Cheung K.M.C., Ngan A.H.W., Chan D., Leung Y.L., Lim C.T. and Luk K.D.K., Correlation between the nano-structure and macro-mechanics of human nucleus pulposus, Combined SICOT/RCOST 2009 Annual Meeting, Pattaya, Thailand, October 29-November 1, 2009.

Sze K.L., Nagy A.N.D.R.A.S., Fosang A.M.A.N.D.A., McCulloch D.A.N.I.E.L., Cheung K.M.C. and Chan D., Poster Presentation: Impact Of Activating Adamts-5 In Cartilage-related Degenerative Disorders , UGC AoE Site Visit . 2010.

Tan Z., Niu B., Tsang K.Y., Melhado I.G., Zhang M., Chan D. and Cheah K.S.E., Global gene expression changes during chondrocyte adaptation to ER stress, 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium, CUHK, Hong Kong, 15 May,. 2010.

Tan Z., Niu B., Chan D. and Cheah K.S.E., Mechanisms Underlying Chondrocyte Reprogramming Surviving ER Stress, 14th Research Postgraduate Symposium, December 2 & 3, 2009, The University of Hong Kong. 2009.

Tang H.C., Yang Y., Chan D. and Cheah K.S.E., Developmental Origins of Osteoblasts in Endochondral Bone Formation, 14th Research Postgraduate Symposium, December 2 & 3, 2009, The University of Hong Kong. 2009.

Tang H.C., Yang L., Tsang K.Y., Chan D. and Cheah K.S.E., Dual origin of osteoblasts in bone formation - terminally differentiated chondrocytes contribute to bone formation in vivo, 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium, CUHK, Hong Kong, 15 May,. 2010.

Tsang K.Y., Chan D., Bateman J.F. and Cheah K.S.E., In vivo cellular adaptation to ER stress: survival strategies with double-edged consequences, J. Cell Science. 2010, 123: 2145-21554.

Tsang K.Y., Cheung K.M.C., Chan D. and Cheah K.S.E., The developmental roles of the extracellular matrix: beyond structure to regulation, Cell Tissue Res. 2010, 339(1): 93-110.

Tsui Y.K., Leung Y.L., Kao R.Y.T., Masuda K., Chan D. and Cheung K.M.C., Identifying therapeutic chemical agents for intervertebral disc degeneration by high throughput screening, 56th Annual Meeting of the Orthopaedic Research Society, New Orleans, Louisiana, March 6-9, 2010.

Tsui Y.K., Cheung K.M.C., Leung Y.L., Chan D. and Kao R.Y.T., Uncovering new compounds for treatment of interveretebral disc degeneration by chemical genetics, 2010.

Wang X., Cheung M.C.H. and Chan D., Molecular consequences of Indian hedgehog mutations in chick neural tube, 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium, CUHK, Hong Kong, 15 May,. 2010.

Wang Y., Lu L., Wu M.H., Sham M.H., Chan D., Cheah K.S.E. and Cheung M.C.H., In vitro generation of Osteo-chondroprogenitors, 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium, CUHK, Hong Kong, 15 May,. 2010.

Yang F., Leung Y.L., Luk K.D.K., Chan D. and Cheung K.M.C., 250. Mesenchymal stem cells arrest intervertebral disc degeneration through chondrocytic differentiation and stimulation of endogenous cells, Molecular Therapy. 2009, 17: 1959-1966.

Yang F., Leung Y.L., Luk K.D.K., Chan D. and Cheung K.M.C., Mesenchymal stem cells arrest intervertebral disc degeneration through chondrocytic differentiation and stimulation of endogenous cells, Molecular Therapy. 2009.

Yee F.Y.A., Cheung K.M.C. and Chan D., Transcriptome and proteome of the intervertebral disc in health and disease, 2010.

Yeung C.W., Cheah K.S.E., Chan D. and Chan B.P., Effects of reconstituted collagen matrix on fates of mouse embryonic stem cells before and after induction for chondrogenic differentiation, 7th Annual Meeting of International Society for Stem Cell Research, Jul 8-11, 2009. Barcelona, Spain.. 2009, 369.

Yeung K.W.K., Lam K.O., Luk K.D.K., Cheung K.M.C., Wu S.L., Hu T., Liu X.Y., Chu C.L., Chu P.K. and Chan D., Long Term Cytocompatibility and In vivo Investigation of Nitrogen Plasma Implanted Shape Memory Alloy. , International Conference on Shape Memory ands Superelastic Technologies (SMST), Pacific Grove, California (16-20May2010). 2010, 104-105.

Researcher : Chan JKM

List of Research Outputs

Zhou Z., Jin G. and Chan J.K.M., Mt1-mmp Regulates Fgf Signaling In Osteogenesis, Gordon Research Conference-Matelloproteinses. Les Diablerets, Switzerland, 2009.

Researcher : Chan KT

List of Research Outputs

Chan K.T., Qi J. and Sham M.H., Multiple coding and non-coding RNAs in the Hoxb3 locus and their spatial expression patterns during mouse embryogenesis, Biochem Biophys Res Commun. 2010, 23:398(2): 153-9.

Researcher : Chan SCL

List of Research Outputs

Chan S.C.L., Expression profiling and epigenetic regulation of Hox genes in cellular models of chondrogenesis, M.Phil Thesis, 2010.

Chan S.C.L. and Sham M.H., Hox Expression Profile and its Epigenetic Regulation during Chondrogenesis, 14th Research Postgraduate Symposium, December 2 & 3, 2009, The University of Hong Kong. 2009.

Researcher : Chan WCW

List of Research Outputs

Lie Y.S., Chan W.C.W., Tsang K.Y., Cheah K.S.E. and Chan D., Impact of intracellular clearance of unfolded proteins on chondrocytes cell fate, 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium, CUHK, Hong Kong, 15 May,. 2010.

Researcher : Chan WY

List of Research Outputs

Cheah K.S.E., Au Y.K., Szeto Y.Y., Wynn S., Chan Y.S., Cheung K.M.C., Chan W.Y., Lovell-Badge R.H., Chan D. and Fritzsch B., Molecular and developmental insights into the pathogenesis of the SOX9Y440Xcampomelic dysplasia mutation, 16th International Society of Developmental Biologists Congress 6-10th September 2009 Edinburgh International Conference Centre, UK . 2009.

Researcher : Cheah KSE

Project Title:	The role of Type II procollagens encoded by alternatively spliced forms of aI(II) procollagen mRNA in cartilage differnetiation and growth
Investigator(s):	Cheah KSE
Department:	Biochemistry
Source(s) of Funding:	Arthritis and Rheumatism Council (ARC) - General Award
Start Date:	12/1993

Abstract:

To gain insight into the function of type IIA procollagen by: a) performing a loss-of function test by introducing into the mouse germ line, a mutation in the [alpha]1(II) collagen gene in which exon 2 is deleted, using gene targeting; determining the phenoypic consequence of reduced levels of expression or failure to express type IIA procollage; generating monoclonal antibodies to the cysteine-rich domain within the aminopropeptide of type IIA procollagen.

Project Title:	Defining the role of the transcription factor Sox9 in skeletal development by tissue-specific gene inactivation
Investigator(s):	Cheah KSE
Department:	Biochemistry
Source(s) of Funding:	UK/Hong Kong Joint Research Scheme
Start Date:	02/1998

Abstract:

To inactivate the mouse Sox9 gene selectively and specifically in developing cartilage tissue in order to understand the role of Sox9 in skeletal formation and the relationship between loss of Sox9 gene function and skeletal malformation.

Project Title:	4th Pan-Pacific Connective Tissues Societies Symposium Abnormal Regulation of Cartilage and Bone Growth in Transgenic Mice Expressing a Collagen X SMCD Mution
Investigator(s):	Cheah KSE
Department:	Biochemistry
Source(s) of Funding:	URC/CRCG - Conference Grants for Teaching Staff
Start Date:	11/1999

Abstract:

N/A

Project Title:	Human Genome Meeting 2001 Genomic Analysis of the Mutant Locus in the Novel Transgenic Mouse Mutant, Yellow Submarine (ysb)
Investigator(s):	Cheah KSE
Department:	Biochemistry
Source(s) of Funding:	URC/CRCG - Conference Grants for Teaching Staff
Start Date:	04/2001

Abstract:

N/A

Project Title:	67th Cold Spring Harbor Symposium on Quantitative Biology - The Cardiovascular System An Essential Role for Type IIA Procollagen in Heart Development
Investigator(s):	Cheah KSE
Department:	Biochemistry
Source(s) of Funding:	URC/CRCG - Conference Grants for Teaching Staff
Start Date:	05/2002

Abstract:

N/A

Project Title:	1st Annual Meeting of the American Society for Matrix Biology Type IIA Procollagen is Essential for Proper Heart Development
Investigator(s):	Cheah KSE
Department:	Biochemistry
Source(s) of Funding:	URC/CRCG - Conference Grants for Teaching Staff
Start Date:	11/2002

Abstract:

N/A

Project Title:	The Gordon Research Conference on Cartilage Biology and Pathology Chondrocytes Survive ER Stress Caused by Unfolded Proteins and Differentiation is Re-programmed
Investigator(s):	Cheah KSE
Department:	Biochemistry
Source(s) of Funding:	URC/CRCG - Conference Grants for Teaching Staff
Start Date:	06/2005

Abstract:

N/A

Project Title:	Genomic approaches to uncover functionally relevant signalling pathways in craniofacial development
Investigator(s):	Cheah KSE, Chan WY, Huang J, Sham MH, Smith DK, Chung SK, Zhou Z
Department:	Biochemistry
Source(s) of Funding:	Collaborative Research Fund (CRF) - Group Research Project
Start Date:	03/2006
Completion Date:	09/2009

Abstract:

To uncover and understand the roles of two sets of transcription factor genes: Hos genes in establishing the complex pattern and structures of the inner ear and middle ear; and Sox9 and Sox10 in the early induction of the inner ear and the later specification of cell fate or type; to generate hypotheses on genetic relationships, molecular interactions and pathways involving Hoxa/b, Six1, Sox9 and Sox10 using simpler model systems such as the worm; to validate the hypotheses on molecular interactions by molecular, biochemical and cellular approaches.

Project Title:	11th Human Genome Meeting Procollagen IIA Facilitates BMP Signaling in Heart Development
Investigator(s):	Cheah KSE
Department:	Biochemistry
Source(s) of Funding:	URC/CRCG - Conference Grants for Teaching Staff
Start Date:	05/2006

Abstract:

N/A

Project Title:	Cold Spring Harbor Laboratory Meeting on Mouse Molecular Genetics Insight into the Molecular Pathogenesis of Campomelic Dysplasia Received By A Conditional SOX9 Mutant Mouse Model
Investigator(s):	Cheah KSE
Department:	Biochemistry
Source(s) of Funding:	URC/CRCG - Conference Grants for Teaching Staff
Start Date:	08/2006

Abstract:

N/A

Project Title:	Development and Reproduction
Investigator(s):	Cheah KSE
Department:	Biochemistry
Source(s) of Funding:	Seed Funding for Strategic Research Theme
Start Date:	09/2008
Completion Date:	08/2011

Abstract:

n/a

Project Title:	Role of Sox2 in the stemness of dermal precursors
Investigator(s):	Cheah KSE
Department:	Biochemistry
Source(s) of Funding:	Seed Funding Programme for Basic Research
Start Date:	03/2010

Abstract:

The skin is the largest organ in the mammalian body, allowing interaction with and providing protection from the surrounding environment. The remarkable regeneration capacity of the skin and its appendages (hair follicles) are attributed to the regulated activation of dedicated stem cell populations1,2. Several distinct populations of stem cells have been identified in rodent skin. Mouse hair follicle stem cells are thought to reside in the hair follicle bulge in outer root sheath (ORS) and are characterized by expression of the CD34 cell-surface marker3. At the start of each hair cycle, signals from dermal papilla (DP) activate these quiescent bulge stem cells which proliferate and migrate down the hair shaft to produce matrix cells. In addition, Blimp14 expressing unipotent progenitor cells which reside in the sebaceous gland (SG) control gland homeostasis and the proliferation of bulge stem cells. Genetic lineages studies have revealed that an additional distinct population of interfollicular epidermal stem cells exists2 and is thought to reside in the basal layer of the epidermis. However, the identity, number and exact location of these “epidermal” stem cells is still unclear. Prof. Freda Miller (University of Toronto Hospital for Sick Children) has demonstrated that dermal-origin stem cells are located in the dermal papilla5. These multipotent stem cells, termed skin-derived precursors (SKPs), have been shown to be able to differentiate into neurons, glia, Schwann cell, smooth muscle cell, adipocyte and skeletal cells in vitro and in vivo6,7,8. Interestingly using a mouse line expressing EGFP reporter in the Sox2 locus they recently found Sox2 expressing cells in the skin and that SKPs sphere formation efficiency is highly increased in the Sox2 positive population. These results implicate Sox2 positive cells as the major source of SKPs5. Sox2, a member of the SoxB1 subfamily of HMG box transcription factors, plays a central role in the development of sensory organs such as retina9, inner ear10 and taste bud11. It is also essential for maintaining several stem cell populations12, for the pluripotent cells of the preimplantation mouse embryo13, and for the embryo-derived stem cells14. It is one of the Yamanaka factors (Sox2, Oct4, Klf4, c-myc) which are required for the induction of the pluripotency of the somatic cells15. We have characterized the expression of Sox2 during hair morphogenesis. In the initiation of hair placode formation (~E14.5), Sox2 is expressed in the dermal condensate of the placode (Fig 1A). After E15.5, Sox2 is expressed in the DP (Fig 1B, D) of the hair follicle and in the epidermal Merkel cells (Fig 1B, C). Surprisingly, we also found that Sox2 is expressed in distinct hair types. It is only expressed in the DP of primary hair and secondary hair but not the tertiary hair (Fig. 2). In our previous study, we have shown that Light Coat and Circling (Lcc) and Yellow Submarine (Ysb), are allelic mouse mutants which display recessive deafness10 and a semi-dominant coat color change from agouti to yellow as a result of loss of black awls, increase in zigzags and alteration of pigmentation pattern in hairs such as an extra yellow band appeared in Lcc mutant16. We have determined that the cause of the inner ear defect in both mutants is failure or impaired ability to establish the prosensory domain and this phenotype is due to the absence and/or down-regulation of Sox2 expression within the inner ear during development10. However the molecular cause of the hair phenotype in these mutants is not known. Since the ear defects are caused by deficiency of Sox2, we tested for Sox2 expression in the DP of both mutants and found that its expression lost is in the skin during hair morphogenesis (Fig 3A, B). We also found premature loss of vibrissae (whiskers) in Lcc/Lcc mutants, suggested that Sox2 expression in the DP is critical for stem cell renewal (Fig 3C-E). Finally in preliminary studies, we found that the proliferation rate of the primary SKPs derived from both Lcc and Ysb mutants is significantly increased (Fig 3F), suggested that the intrinsic dermal stem cell property is altered with down-regulation of Sox2. These mutants, therefore, provide an opportunity to study the roles of Sox2 in hair follicle development and DP stem cell maintenance in both pre-natal and post-natal stages. Our hypothesis is that the Sox2 expressing cells in the DP of primary and secondary hair may be dermal stem cells and that Sox2 has an important role in their maintenance. Since Sox2 is a transcription factor the likely change in property of the SKPs would be in the expression of genes regulated by Sox2. In order to build a hypothesis for the mechanism by which loss of Sox2 in the DP and SKPs causes premature loss of whisker hair it is essential to determine the impact of loss of Sox2 on the gene expression profile of SKPs isolated from Lcc/Lcc. The objective of this project is to isolate SKPs from Lcc/Lcc DP and compare the global gene expression profiles of these mutant cells with wild-type SKPs.

List of Research Outputs

Cheah K.S.E., Alleviating ER Stress in Chondrocytes: Molecular Strategies and the Developmental Cost of Recovery., Gordon Research Conference on Collagen, New London, USA, 19-24 July 2009.

Cheah K.S.E., Wang C., Tan Z., Melhado I.G., Niu B., Tsang K.Y., Zhang M...Q..., Ron D... and Chan D., Alleviating ER stress in chondrocytes: survival strategies with double-edged developmental consequences , 43rd Annual Meeting for the Japanese Society of Developmental Biologists Jointly Sponsored by the Asia-Pacific Developmental Biology Network, Kyoto, Japan, 20 June 2010.

Cheah K.S.E., Cell fate determination in skeletal development and growth., 25th Ernst Klenk symposium in Molecular Medicine, Cologne, Germany, 4-6 October 2009.

Cheah K.S.E., Wang C., Tan Z., Melhado I.G., Niu B., Tsang K.Y., Zhang M...Q..., Ron D. and Chan D., Genetic and genomic analyses of chondrocyte adaptation to ER stress, The Genetics Society Spring Meeting “Mouse Genetics – Think Globally, Act Locally” The Wellcome Trust Conference Centre, Hinxton, UK, 23 April 2010 . 2010.

Cheah K.S.E., Au Y.K., Szeto Y.Y., Wynn S., Chan Y.S., Cheung K.M.C., Chan W.Y., Lovell-Badge R.H., Chan D. and Fritzsch B., Molecular and developmental insights into the pathogenesis of the SOX9Y440Xcampomelic dysplasia mutation, 16th International Society of Developmental Biologists Congress 6-10th September 2009 Edinburgh International Conference Centre, UK . 2009.

Cheah K.S.E., Molecular and developmental insights into the pathogenesis of the Sox9Y44X campomelic dysplasia mutation, HKU-PUMC Joint Research Meeting, Beijing, China, 11 June 2010. 2010.

Cheah K.S.E., Molecular and developmental insights into the pathogenesis of the Sox9^Y44X campomelic dysplasia mutation, HKU-PUMC Joint research meeting, Beijing, China. 2010.

Choi M.Y., Chan C.C.Y., Chan D., Luk K.D.K., Cheah K.S.E. and Tanner J.A., Biochemical consequences of sedlin mutations that cause spondyloepiphyseal dysplasia tarda, Biochemical Journal. Great Britain, Biochemical Society, 2009, 423: 233-242.

Chu K.H., Zhang M., Wong E.Y.M., Szeto Y.Y., Chan Y.S., Cheah K.S.E. and Sham M.H., Best Poster Award: Sox10 mutation affects cochleo-vestibular ganglion gliogenesis, 43rd Annual Meeting for the Japanese Society of Developmental Biologists, Kyoto, Japan, 20-23 June, 2010.. 2010.

Chu K.H., Zhang M., Wong E.Y.M., Szeto Y.Y., Chan Y.S., Cheah K.S.E. and Sham M.H., Best Poster Award: Sox10 mutation affects gliogenesis of the cochleo-vestibular ganglion, 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium, 15 May 2010. 2010.

Chu K.H., Zhang M., Wong E.Y.M., Szeto Y.Y., Chan Y.S., Cheah K.S.E. and Sham M.H., Sox10 mutation affects cochleo-vestibular ganglion gliogenesis, 43rd Annual Meeting for the Japanese Society of Developmental Biologists, Kyoto, Japan, 20-23 June. 2010.

Chu K.H., Zhang M., Wong E.Y.M., Szeto Y.Y., Chan Y.S., Cheah K.S.E. and Sham M.H., Sox10 mutation affects gliogenesis of the cochleo-vestibular ganglion, 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium, 15 May 2010.

Gao Y., Leung W.L., Wong S.Y.Y., Tam P.P.L., Chan D. and Cheah K.S.E., Type IIA Procollagen: A Positive Regulator of Nodal Signaling During Early Vertebrate Embryogenesis, 14th Research Postgraduate Symposium, December 2 & 3, 2009, The University of Hong Kong. 2009.

Gao Y., Leung W.L., Wong Y.Y., Chan D., Tam P.P.L. and Cheah K.S.E., Type IIA procollagen: A positive regulator of nodal signaling during embryogenesis, 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium, CUHK, Hong Kong, 15 May. 2010.

Guo S., Zhou J., Gao B., Hu J., Wang H., Meng J., Zhao X., Ma G., Lin C., Xiao Y., Tang W., Zhu X., Cheah K.S.E., Feng G., Chan D. and He L., Missense mutations in IHH impair Indian Hedgehog signaling in C3H10T1/2 cells: Implications for brachydactyly type A1, and new targets for Hedgehog signaling, Cell Mol Biol Lett. 2009, 15(1): 153-76.

Ho B.S., Cheng C.C.W., Leung K.K.H. and Cheah K.S.E., The role of Sox2 and Sox8 in mouse hair development, 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium, CUHK, Hong Kong, 15 May,. 2010.

Kao Y.P.P., Chan D., Cheah K.S.E., Cheung K.M.C., Ho D.W.H., Karppinen J., Leong J.C.Y., Yip S.P., Song Y. and Sham P.C., Genome-wide Association Study of Degenerative Disc Disease (DDD), 14th Research Postgraduate Symposium, December 2 & 3, 2009, The University of Hong Kong. 2009.

Lee K.S., Lam T.K., Song Y., Cheah K.S.E., Cheung K.M.C. and Chan D., In vivo study of Asporin, a genetic risk factor for Osteoarthritis and degenerative disc disease, 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium, CUHK, Hong Kong, 15 May,. 2010.

Leung Y.L., Gao B., Melhado I.G., Leung K.K.H., Wynn S.L., Chan D. and Cheah K.S.E., Sox9 mediates gene expression specificity in growth plate chondrocytes via concomitant positive and negative regulation, 55th Annual Meeting of the Orthopaedic Research Society, New Orleans, USA. 2010.

Li J., Lee Y.F. and Cheah K.S.E., Identification of regulatory elements directing control of Sox2 expression in mouse inner ear development, 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium, CUHK, Hong Kong, 15 May,. 2010.

Lie Y.S., Chan W.C.W., Tsang K.Y., Cheah K.S.E. and Chan D., Impact of intracellular clearance of unfolded proteins on chondrocytes cell fate, 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium, CUHK, Hong Kong, 15 May,. 2010.

Lu L., Chan D., Cheah K.S.E. and Cheung M.C.H., Epigenetic Reprogramming of Fibroblasts into Osteo-chondroprogenitors, 14th Research Postgraduate Symposium, December 2 & 3, 2009, The University of Hong Kong. 2009.

Lu L., Cheah K.S.E., Chan D. and Cheung M.C.H., Epigenetic reprogramming of fibroblasts into osteo-chondroprogenitors, 43rd Annual Meeting for the Japanese Society of Developmental Biologists Kyoto, Japan, 20 June 2010 . 2010.

Mak A.C.Y., Szeto Y.Y., Fritzsch B. and Cheah K.S.E., Differential and overlapping expression pattern of SOX2 and SOX9 in inner ear development, Gene Expression Patterns. 2009, 9: 444-453.

Shao D., Baker M.D., Abrahamsen B., Rugiero F., Malik-Hall M., Poon W.Y., Cheah K.S.E., Yao K.M., Wood J.N. and Okuse K., A multi PDZ-domain protein Pdzd2 contributes to functional expression of sensory neuron-specific sodium channel Nav1.8, Molecular and Cellular Neuroscience. Elsevier, 2009, 42: 219-225.

Song Y., Tang L.F., Cheung C.L., Sham P.C., McClurg P., Smith D.K., Tanner J.A., Su A.I., Cheah K.S.E. and Kung A.W.C., Genome-wide haplotype association mapping in mice identifies a genetic variant in CER1 associated with bone mineral density and fracture in southern Chinese women, The American Society of Human Genetics 59th Annual Meeting, Honolulu, Hawaii. 2009.

Tan Z., Niu B., Tsang K.Y., Melhado I.G., Zhang M., Chan D. and Cheah K.S.E., Global gene expression changes during chondrocyte adaptation to ER stress, 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium, CUHK, Hong Kong, 15 May,. 2010.

Tan Z., Niu B., Chan D. and Cheah K.S.E., Mechanisms Underlying Chondrocyte Reprogramming Surviving ER Stress, 14th Research Postgraduate Symposium, December 2 & 3, 2009, The University of Hong Kong. 2009.

Tang H.C., Yang Y., Chan D. and Cheah K.S.E., Developmental Origins of Osteoblasts in Endochondral Bone Formation, 14th Research Postgraduate Symposium, December 2 & 3, 2009, The University of Hong Kong. 2009.

Tang H.C., Yang L., Tsang K.Y., Chan D. and Cheah K.S.E., Dual origin of osteoblasts in bone formation - terminally differentiated chondrocytes contribute to bone formation in vivo, 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium, CUHK, Hong Kong, 15 May,. 2010.

Thomsen M.K., Ambroisine L., Wynn S., Cheah K.S.E., Foster C.S., Fisher G., Berney D.M., Moller H., Reuter V.E., Scardino P., Cuzick J., Ragavan N., Singh P.B., Martin F.L., Butler C.M., Cooper C.S. and Swain A., SOX9 elevation in the prostate promotes proliferation and cooperates with PTEN loss to drive tumor formation., Cancer Research. 2010, 70(3): 979-87.

Tsang K.Y., Chan D., Bateman J.F. and Cheah K.S.E., In vivo cellular adaptation to ER stress: survival strategies with double-edged consequences, J. Cell Science. 2010, 123: 2145-21554.

Tsang K.Y., Cheung K.M.C., Chan D. and Cheah K.S.E., The developmental roles of the extracellular matrix: beyond structure to regulation, Cell Tissue Res. 2010, 339(1): 93-110.

Tsang S.W., Shao D., Cheah K.S.E., Okuse K., Leung P.S. and Yao K.M., Increased basal insulin secretion in Pdzd2-deficient mice, Mol Cell Endocrinol. 2010, 315(1-2): 263-70.

Wang Y., Lu L., Wu M.H., Sham M.H., Chan D., Cheah K.S.E. and Cheung M.C.H., In vitro generation of Osteo-chondroprogenitors, 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium, CUHK, Hong Kong, 15 May,. 2010.

Yang S., Agrawal K.R., Lam T.W., Sham P.C., Cheah K.S.E. and Wang J.J., Novel Profile Hidden Markov Model to Predict MicroRNAs and their Targets Simutaneously, 14th Research Postgraduate Symposium, The University of Hong Kong, December 2-3. 2009.

Yang S., Agrawal K.R., Lam T.W., Sham P.C., Cheah K.S.E. and Wang J.J., Novel Profile Hidden Markov Model to predict microRNAs in Degenerative Disc Disease. University Grants Council site visit for Area of Excellence Programme "Developmental Genomics & Skeletal Research". January 25, 2010.

Yeung C.W., Cheah K.S.E., Chan D. and Chan B.P., Effects of reconstituted collagen matrix on fates of mouse embryonic stem cells before and after induction for chondrogenic differentiation, 7th Annual Meeting of International Society for Stem Cell Research, Jul 8-11, 2009. Barcelona, Spain.. 2009, 369.

Researcher : Cheng CCW

Project Title:	Role of Sox2 in Pituitary Development
Investigator(s):	Cheng CCW, Cheah KSE
Department:	Biochemistry
Source(s) of Funding:	Small Project Funding
Start Date:	01/2009

Abstract:

The pituitary gland exerts its pivotal roles in homeostatic regulation of vertebrates by signaling to peripheral organs. In all vertebrates studied, pituitary gland consists of three sections (anterior, intermediate and posterior lobes). The mature anterior lobe contains five hormone-producing cell types, including corticotropes secreting adrenocorticotropin (ACTH); thyrotropes secreting thyroid-stimulating hormone (TSH); gondadotropes secreting luteinizing hormone (LH) and follicle-stimulating hormone (FSH); somatotropes secreting growth hormone (GH); and lactotropes secreting prolactin (PRL). The intermediate lobe contains melanotropes secreting melanocyte-stimulating hormone (MSH) and the posterior lobe can produce antidiuretic hormone and oxytocin. The anterior and intermediate lobes of the pituitary first appear at embryonic day 8 (E8) in the mouse as a thickening of oral ectoderm that invaginates to form the structure known as Rathke’s pouch (for review, Burrows et al., 1999). Rathke’s pouch separates from the oral ectoderm by E12.5. Cells on the anterior side of the pouch undergo proliferation to generate the anterior lobe, whereas cells on the opposite side form the intermediate lobe. The posterior lobe develops concurrently from neural ectoderm. SOX2, a member of the SOXB1 subfamily of HMG box transcription factors, is essential for maintaining several stem cell populations (Penvy & Placzek, 2005), for the pluripotent cells of the preimplantation mouse embryo (Avilion et al., 2003), and for the embryo-derived stem cell (Boiani & Scholer, 2005). It is also required for the development of sensory organs such as retina (Taranova et al., 2006), inner ear (Kiernan et al., 2005) and taste bud (Okubo et al., 2006). Sox2 RNA is first detected in some cells at morula stages at E2.5 and in within the ICM of blastocysts at E3.5 (Avilion et al., 2003). Expression persists throughout the epiblast, but by mid-late-streak stages (E7.0–7.5), it becomes restricted to presumptive neuroectoderm in the anterior, while it is excluded from the posterior, including cells ingressing through the primitive streak. At E11.5, Sox2 is expressed in the presumptive hypothalamus and is ubiquitously expressed throughout the primordium of the anterior and intermediate pituitary, Rathke’s pouch (Kelberman et al., 2006). By E16.5, cells in the pituitary are differentiating ventrally, and SOX2 is still widely expressed in the differentiated cells by immunohistochemistry (Fauquier et al., 2008). By E18.5, SOX2 is mostly expressed in the dorsal proliferative zone of the developing pituitary and in scattered cells of the anterior lobe. In adult pituitary glands, SOX2 expressing cells (representing 3–5% of the cells in the anterior lobe) are present in a concentrated layer lining the pituitary cleft and are scattered throughout the parenchyma. In our previous study, we have shown that Light Coat and Circling (Lcc) and Yellow Submarine (Ysb), are allelic mouse mutants which display recessive deafness (Kiernan et al., 2005) and a semi-dominant coat color change from agouti to yellow as a result of loss of black awls, increase in zigzags and alteration of pigmentation pattern in hairs such as an extra yellow band appeared in Lcc mutant (Dong et al., 2002). We have demonstrated that the inner ears of both mutants fail to establish the prosensory domain and this phenotype is due to the absence and/or down-regulation of SOX2 expression within the inner ear during development. We recently also found SOX2 is down-regulated in the intermediate lobe and posterior lobe of developing pituitary in these two mutants (Fig. 1). These mutants, therefore, provide an opportunity to study the role of Sox2 in pituitary in both pre-natal and post-natal stages. The specific hypothesis of this proposal is that Sox2 is a major regulator for the differentiation of hormone producing cells types in the pituitary. That hypothesis is based on the following observations. First, it has been shown that heterozygous mutations within SOX2 in humans have been associated with bilateral anophthalmia or severe microphthalmia, in addition to hypopituitarism characterized by anterior pituitary hypoplasia and gonadotropin deficiency (hypogonadotropic hypogonadism) with genital abnormalities (Sato et al., 2007; Kelberman et al, 2008). Sox2 -/- null embryos die shortly after implantation; however Sox2 heterozygous mice appear relatively normal, but about one third show perinatal lethality and others show a reduction in size and male fertility (Avilion et al., 2003). Analysis of heterozygous mutant mice showed a variable hypopituitary phenotype affecting the size and shape of the pituitary gland and disruption of the endocrine homeostasis (Kelberman et al, 2006). Second, in nonadherent pituispheres culture of adult anterior pituitary, SOX2 expression is detected in the self-renewing, pluripotency progenitors (Fauquier et al., 2008). In this culture system, SOX2 expressing cells have been shown to be able to differentiate to corticotropes, thyrotropes, gondadotropes, somatotropes and lactotropes. Third, MSH (produced in intermediate lobe) induces eumelanin production, resulting in the subterminal band of phaeomelanin often visible in mammalian fur and regulates the hair pigmentation (for review, Voisey and van Daal, 2002). It is produced in the intermediate lobe of pituitary and is a critical factor for hair pigmentation (Burchill et al., 1993).We have found defects in hair pigmentation in both Ysb and Lcc mutants (Dong et al., 2002) and this phenotype is likely related to the alteration of MSH level from the intermediate lobe of the pituitary. The objectives of this study are: 1) To determine and characterize the developmental timing and specificity of down-regulation of SOX2 in the pituitaries in Lcc and Ysb mutants. 2) To examine the effects of the down-regulation of SOX2 on differentiation of pituitary hormone-producing cells of these two mutants. 3) To compare the progenitor potential and the pluripotency of the pituispheres generated from these SOX2 down-regulated pituitaries to their wild-type counterpart by using pituispheres culture system.

List of Research Outputs

Ho B.S., Cheng C.C.W., Leung K.K.H. and Cheah K.S.E., The role of Sox2 and Sox8 in mouse hair development, 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium, CUHK, Hong Kong, 15 May,. 2010.

Researcher : Cheng LYL

Project Title:	A concordance study of the importance of autosomal short tandem repeat (STR) and Y-chromosome STR in the Chinese community
Investigator(s):	Cheng LYL, Tan-Un KC
Department:	Biochemistry
Source(s) of Funding:	Small Project Funding
Start Date:	11/2002

Abstract:

In recent years, Short Tandem repeat (STR) and microsatellite analyses offer invaluable evidences in forensic science casework in court. Despite the well established database in the US forensic system, an Asian population database urgently needed to be established. In this research study, we use the STR markers that suit the American Combined DNA index system (CODIS) required by FBI to analyse Asian Chinese samples. Hence, the frequency of the allele for each locus can be calculated statistically, and stored for the use of the Chinese community.

Researcher : Cheng MH

List of Research Outputs

Cheng M.H., The novel mouse γ A-crystallin mutation leads to misfolded protein aggregate and cataract, MPhil Thesis. 2009.

Tam C.N., Cheng M.H., Tsang S.L., Yam G.H.F., Pang C.P. and Sham M.H., Best Poster Award: Unfolded Protein Response Leading to Cataratogenesis in a Microphthalmia Cataract Mouse Mutant., 14th Research Postgraduate Symposium, December 2 & 3, 2009, The University of Hong Kong. 2009.

Tam C.N., Cheng M.H., Tsang S.L., Yam G...H...F..., Pang C...P... and Sham M.H., Best Poster Award: Unfolded protein response leading to cataractogenesis in a microphthalmia cataract mouse mutant., 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium, 15 May 2010. . 2010.

Tam C.N., Cheng M.H., Tsang S.L., Yam G.H.F., Pang C.P. and Sham M.H., Unfolded Protein Response Leading to Cataratogenesis in a Microphthalmia Cataract Mouse Mutant, 14th Research Postgraduate Symposium, December 2 & 3, 2009, The University of Hong Kong. 2009.

Tam C.N., Cheng M.H., Tsang S.L., Yam G...H...F..., Pang C...P... and Sham M.H., Unfolded protein response leading to cataractogenesis in a microphthalmia cataract mouse mutant, 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium, 15 May 2010. . 2010.

Tam C.N., Cheng M.H., Tsang S.L., Yam G.H.F., Pang C.P. and Sham M.H., Unfolded protein response leading to cataractogenesis in a microphthalmia cataract mouse mutant, Mech. Dev. . 2009-09-06, 2009, 126: S128-129.

Researcher : Cheng MH

List of Research Outputs

Cheng M.H., The novel mouse γ A-crystallin mutation leads to misfolded protein aggregate and cataract, MPhil Thesis. 2009.

Researcher : Cheung MCH

Project Title:	Investigating the role of SoxE genes in tumor metastasis
Investigator(s):	Cheung MCH
Department:	Biochemistry
Source(s) of Funding:	Seed Funding Programme for Basic Research
Start Date:	10/2008
Completion Date:	10/2009

Abstract:

Neural crest cells (NCCs) are generated at the border between the neural plate and nonneural ectoderm. These cells initiate a distinct program of gene expression, undergo an epithelial-mesenchymal transition (EMT) and delaminate from the neuroepithelium. Our overexpression analysis in chick by in ovo electroporation together with examination of mutant mice showed that the proper specification of the trunk NCCs requires concomitant activity of Sox9, member of the SoxE subgroup of HMG-containing transcription factor, Snail2, zinc-finger transcription factor and FoxD3, winged-helix transcription factor. Sox9 is sufficient to promote NC-like differentiation in neural tube progenitors at the expense of central nervous system neuronal differentiation but requires Snail2 activity to trigger NC delamination with EMT-like characteristics in which cells undergo dramatic changes in cell shape, loss of epithelial conformation and the acquisition of cell motility. However, Snail2 expression alone is not sufficient to induce NC formation and an EMT. In Sox9 mutant embryos, trunk NCCs are still specified and are able to undergo EMT, yet they die soon after the onset of migration. Consistent with this, neuroepithelial cells forced to undergo EMT by expressing RhoB, the small GTPase, fail to migrate and to survive, whereas acquisition of a NC phenotype by expression Sox9 protect RhoB expression cells from apoptosis, suggesting that the inappropriate initiation of an EMT is not compatible with the survival of neural epithelial cells. These data indicate that Sox9 acts to provide the competence for NC to undergo an EMT and is required for trunk NCCs survival. While co-expression of Sox9 and Snail2 is sufficient to induce both NC differentiation and some aspects of EMT in neuroepithelial cells, FoxD3 not only promotes NC-like differentiation process similar to Sox9 but also regulates the expression of cell-cell adhesion molecules required for NC migration. Together, these data suggest a combinatorial role of different families of transcription factors expressed by prospective trunk NCCs, which control and coordinate NC induction, EMT and survival. Our studies demonstrated an integrated transcriptional control of neural crest identity with features of an EMT, which is considered to be an in vivo correlate with metastatic transformation and acquisition of invasive behavior by tumor cells. Previously, expression of Snail1/Snail2 in tumors has been associated with increased invasiveness, and the induction of Snail1/Snail2 in cell line models has been demonstrated to enhance their metastatic characteristics. Similarly, our preliminary studies by quantitative real-time PCR (qRT-PCR) on several epithelial tumor-derived cell lines showed that high level of SoxE transcripts were frequently detected in cells that were mesenchymal, highly invasive and metastatic, suggesting a potential role of SoxE proteins in mediating an EMT for an epithelial tumor cell to progress from noninvasive to invasive state. Together, these data raise the question of whether the induction of epithelial tumor cells invasion by Snail1/Snail2 expression is mediated through SoxE function. It is also possible that SoxE proteins activate Snail family members to induce the invasive properties of tumor cells since Snail2 was found to be one of the direct downstream targets of Sox9 during NC development. Furthermore, our previous findings revealing a competence role of SoxE proteins for neural crest to successfully complete an EMT might imply to have a similar function for tumor cells to undergo transformation. To test these possibilities, our objectives are: 1. To investigate whether there is an epistatic relationship between SoxE and Snail transcription factors to mediate the transformation of epithelial tumors into invasive carcinomas; 2. To investigate whether SoxE proteins are essential for tumor metastasis

Project Title:	16th International Society for Developmental Biology Congress 2009 Regulation of Sox9 Function by SUMOylation in Avian Neural Crest Development
Investigator(s):	Cheung MCH
Department:	Biochemistry
Source(s) of Funding:	URC/CRCG - Conference Grants for Teaching Staff
Start Date:	09/2009
Completion Date:	09/2009

Abstract:

N/A

List of Research Outputs

Cheung M.C.H., Chau K.H.B., Chan A.Y.L., Ng K.T., Wu M.H. and Briscoe J.A.M.E.S., Regulation of Sox9 Function by SUMOylation in avian neural crest development, In: Shinichi Aizawa Maria Leptin Nancy Papalopulu Claudio D Stern Dider Stainer Patrick Tam, Mechanisms of Development 16th International Society of Developmental Biologists Congress 2009 Book of Abstracts. Elsevier, 2009, 126: S308.

Cheung M.C.H., Chau K.H.B., Ng A., Chan A. and Briscoe J., Regulation of Sox9 function by SUMOylation in Avian Neural Crest Development, Mechanisms of Development. Elsevier, 2009, 126: S308.

Kam K.M., Lui V.C.H., Cheung M.C.H. and Tam P.K.H., Expression of engrailed-Hoxb5 transcriptional repressor by Wnt1-Cre produces neurocristopathies of pigmentation and enteric nervous system defects in mice., 43rd Annual Meeting for the Japanese Society of Developmental Biologists Kyoto, Japan, 20 June 2010 . 2010.

Kam K.M., Lui V.C.H., Cheung M.C.H. and Tam P.K.H., Expression of engrailed-Hoxb5 transcriptional repressor by Wnt1-Cre produces neurocristopathies of pigmentation and enteric nervous system defects in mice, 43rd Annual Meeting for the Japanese Society of Developmental Biologists, Jointly Sponsored by the Asia-Pacific Developmental Biology Network, Kyoto, Japan, 20-23 June. 2010.

Kwok S.T.C., Wu M.H., Herve A., Nieto M.A. and Cheung M.C.H., The role of SoxE transcription factors in melanoma development, 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium, CUHK, Hong Kong, 15 May. 2010.

Lu L., Chan D., Cheah K.S.E. and Cheung M.C.H., Epigenetic Reprogramming of Fibroblasts into Osteo-chondroprogenitors, 14th Research Postgraduate Symposium, December 2 & 3, 2009, The University of Hong Kong. 2009.

Lu L., Cheah K.S.E., Chan D. and Cheung M.C.H., Epigenetic reprogramming of fibroblasts into osteo-chondroprogenitors, 43rd Annual Meeting for the Japanese Society of Developmental Biologists Kyoto, Japan, 20 June 2010 . 2010.

Wang X., Cheung M.C.H. and Chan D., Molecular consequences of Indian hedgehog mutations in chick neural tube, 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium, CUHK, Hong Kong, 15 May,. 2010.

Wang Y., Lu L., Wu M.H., Sham M.H., Chan D., Cheah K.S.E. and Cheung M.C.H., In vitro generation of Osteo-chondroprogenitors, 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium, CUHK, Hong Kong, 15 May,. 2010.

Researcher : Cheung YW

List of Research Outputs

Cheung Y.W. and Tanner J.A., Aptamers against P. falciparum lactate dehydrogenase as a new approach to malaria diagnosis. , CUHK Croucher Advanced Study Institute “Structure-Based Screening and Design of Ligands for Protein Targets” . 2009.

Cheung Y.W. and Tanner J.A., Improving Method for Malaria Detection - An Aptamer Approach, 14th Research Postgraduate Symposium, December 2 & 3, 2009, The University of Hong Kong. 2009.

Researcher : Cheung YW

List of Research Outputs

Cheung Y.W. and Tanner J.A., Improving Method for Malaria Detection - An Aptamer Approach, 14th Research Postgraduate Symposium, December 2 & 3, 2009, The University of Hong Kong. 2009.

Researcher : Chin KT

List of Research Outputs

Chan C.P., Mak T.Y., Chin K.T., Ng I.O.L. and Jin D., N-linked glycosylation is required for optimal proteolytic activation of membrane-bound transcription factor CREB-H., J. Cell Sci.. The Company of Biologists, 2010, 123: 1438-1448.

Chan C.P., Mak T.Y., Chin K.T. and Jin D., Requirement of N-linked Glycosylation in the Luminal Domain for Optimal Proteolytic Activation of Liver-enriched transcription Factor CREB-H, 14th Research Postgraduate Symposium, December 2 & 3, 2009, The University of Hong Kong. 2009.

Researcher : Choi MY

List of Research Outputs

Choi M.Y., Chan C.C.Y., Chan D., Luk K.D.K., Cheah K.S.E. and Tanner J.A., Biochemical consequences of sedlin mutations that cause spondyloepiphyseal dysplasia tarda, Biochemical Journal. Great Britain, Biochemical Society, 2009, 423: 233-242.

Researcher : Chu KH

List of Research Outputs

Chu K.H., Asia-Pacific Developmental Biology Network Student Travel Award, 43rd Annual Meeting for the Japanese Society of Developmental Biologists, Kyoto, Japan.. 2010.

Chu K.H., Zhang M., Wong E.Y.M., Szeto Y.Y., Chan Y.S., Cheah K.S.E. and Sham M.H., Best Poster Award: Sox10 mutation affects cochleo-vestibular ganglion gliogenesis, 43rd Annual Meeting for the Japanese Society of Developmental Biologists, Kyoto, Japan, 20-23 June, 2010.. 2010.

Chu K.H., Zhang M., Wong E.Y.M., Szeto Y.Y., Chan Y.S., Cheah K.S.E. and Sham M.H., Best Poster Award: Sox10 mutation affects gliogenesis of the cochleo-vestibular ganglion, 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium, 15 May 2010. 2010.

Chu K.H., Zhang M., Wong E.Y.M., Szeto Y.Y., Chan Y.S., Cheah K.S.E. and Sham M.H., Sox10 mutation affects cochleo-vestibular ganglion gliogenesis, 43rd Annual Meeting for the Japanese Society of Developmental Biologists, Kyoto, Japan, 20-23 June. 2010.

Chu K.H., Zhang M., Wong E.Y.M., Szeto Y.Y., Chan Y.S., Cheah K.S.E. and Sham M.H., Sox10 mutation affects gliogenesis of the cochleo-vestibular ganglion, 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium, 15 May 2010.

Researcher : Chu KH

List of Research Outputs

Chu K.H., Asia-Pacific Developmental Biology Network Student Travel Award, 43rd Annual Meeting for the Japanese Society of Developmental Biologists, Kyoto, Japan.. 2010.

Researcher : Chun CS

List of Research Outputs

Chun C.S., REV7-mediated polyubiquitination and degradation of human REV1, PhD Thesis. 2009.

Chun C.S. and Jin D., Ubiquitin-dependent regulation of translesion polymerases., Biochem Soc Trans. 2010, 38: 110-115.

Researcher : Cui J

List of Research Outputs

Cui J., Wang Z., Cheng Q.N., Lin R., Zhang X., Leung P.S., Copeland N.G., Jenkins N.A., Yao K.M. and Huang J., Targeted Inactivation of KIF5B in Pancreatic Leads to Insulin Secretory Deficiency and Increased b-Cells Mass in vivo, 14th Research Postgraduate Symposium, December 2 & 3, 2009, The University of Hong Kong. 2009.

Researcher : Danchin ALM

List of Research Outputs

Miao Y., Danchin A.L.M. and Huang J., Visualizing the Proteome Escherichia coli by Recombineering Technology, 14th Research Postgraduate Symposium, December 2 & 3, 2009, The University of Hong Kong. 2009.

Researcher : Gao B

List of Research Outputs

Guo S., Zhou J., Gao B., Hu J., Wang H., Meng J., Zhao X., Ma G., Lin C., Xiao Y., Tang W., Zhu X., Cheah K.S.E., Feng G., Chan D. and He L., Missense mutations in IHH impair Indian Hedgehog signaling in C3H10T1/2 cells: Implications for brachydactyly type A1, and new targets for Hedgehog signaling, Cell Mol Biol Lett. 2009, 15(1): 153-76.

Leung Y.L., Gao B., Melhado I.G., Leung K.K.H., Wynn S.L., Chan D. and Cheah K.S.E., Sox9 mediates gene expression specificity in growth plate chondrocytes via concomitant positive and negative regulation, 55th Annual Meeting of the Orthopaedic Research Society, New Orleans, USA. 2010.

Researcher : Gao Y

List of Research Outputs

Gao Y., Leung W.L., Wong S.Y.Y., Tam P.P.L., Chan D. and Cheah K.S.E., Type IIA Procollagen: A Positive Regulator of Nodal Signaling During Early Vertebrate Embryogenesis, 14th Research Postgraduate Symposium, December 2 & 3, 2009, The University of Hong Kong. 2009.

Gao Y., Leung W.L., Wong Y.Y., Chan D., Tam P.P.L. and Cheah K.S.E., Type IIA procollagen: A positive regulator of nodal signaling during embryogenesis, 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium, CUHK, Hong Kong, 15 May. 2010.

Researcher : Guan XY

Project Title:	49th Annual Meeting of the American Society of Human Genetics The Association of Chromosome 8p Deletion and Tumor Metastasis in Human Hepatocellular Carcinoma
Investigator(s):	Guan XY
Department:	Clinical Oncology
Source(s) of Funding:	URC/CRCG - Conference Grants for Teaching Staff
Start Date:	10/1999

Abstract:

N/A

Project Title:	51st Annual Meeting of the American Society of Human Genetics Loss of Tyrosine Aminotransferase may Contribute to the Development of Hepatocellular Carcinoma
Investigator(s):	Guan XY
Department:	Clinical Oncology
Source(s) of Funding:	URC/CRCG - Conference Grants for Teaching Staff
Start Date:	10/2001

Abstract:

N/A

Project Title:	Characterization of role and mechanism of eIF-5A2 in tumor metastasis
Investigator(s):	Guan XY
Department:	Clinical Oncology
Source(s) of Funding:	General Research Fund (GRF)
Start Date:	10/2007

Abstract:

To investigate the roles eIF-5A2 in tumor invasion and metastasis; to define the role of DHPS in eIF-5A2-associated metastasis; to identify other metastasis related genes in DHPS/eIF-5A2/metastasis pathway.

Project Title:	Evaluation of an Oxygen Therapeutic (IKOR-2084) in the Enhancement of Chemotherapy and Radiotherapy in Rodent Cancer Models
Investigator(s):	Guan XY, Man K, Kwong DLW
Department:	Clinical Oncology
Source(s) of Funding:	Matching Grant for Joint Research
Start Date:	09/2008
Completion Date:	03/2010

Abstract:

(1) To determine the efficacy of IKOR-2084 in the delivery of oxygen to tumor area; (2) To evaluate the efficacy of IKOR-2084 in the sensitization of chemo-therapy with a rat orthotopic liver cancer model; (3) To evaluate the efficacy of IKOR-2084 in the sensitization of radiation therapy with a mouse xenografic head and neck cancer model.

Project Title:	Characterization of anti-apoptotic mechanism of ALC1 in the development of hepatocellular carcinoma
Investigator(s):	Guan XY
Department:	Clinical Oncology
Source(s) of Funding:	Small Project Funding
Start Date:	10/2008

Abstract:

Research Background: Hepatocellular carcinoma (HCC) is the sixth most common human cancer in the world, and its prognosis is extremely poor [1]. Significant efforts have been directed towards charting the genetic alterations in HCC in order to further understanding the genetic basis of the disease and identify new therapeutic targets. Amplification of 1q21 is one of the most frequent genetic alterations in HCC; it has been detected in over 50% of HCC patients [2, 3], strongly suggesting the existence of an oncogene within this region. Recently, we isolated a candidate oncogene ALC1 within the 1q21 region by hybrid selection using microdissected DNA from this region [4]. In that work, we found that ALC1 has strong oncogenic activities, including promoting cell proliferation, increasing colony formation in soft agar, inducing tumor formation in nude mice, and inhibiting cell apoptosis [4]. These data strongly suggest that ALC1 is the target oncogene responsible for the 1q21 amplification event and that it plays important role in HCC development. To further explore the molecular mechanism of ALC1 in HCC pathogenesis, a yeast two-hybrid assay was used to identify proteins that interact with ALC1. One protein, Nur77, which is a unique transcriptional factor belonging to the orphan nuclear receptor superfamily was isolated. The interaction between ALC1 and Nur77 has been confirmed by co-immunoprecipitation (unpublished data). Similar to the death signaling that occurs when p53 interacts with anti- and pro-apoptotic Bcl-2 family members in mitochondria, Nur77 directly targets mitochondria and initiates cell apoptosis by binding to Bcl-2 [5]. Several studies have demonstrated that Nur77 protein translocates from the nucleus to the mitochondria in response to pro-apoptotic stimuli [6, 7]. In the mitochondria, Nur77 interacts with Bcl-2 and induces the latter to adopt a pro-apoptotic conformation that triggers downstream apoptotic events, including cytochrome c (Cyt c) release, activation of Apaf-1 and Caspase-9 (Casp 9), and cleavage of pro-Caspase-3 [7]. Escaping apoptosis is one of the major mechanisms of cancer progression, because it extends cell survival and allows for the accumulation of genetic instability and mutations. Therefore, these findings prompted us to assume that the anti-apoptotic activity of ALC1 may involve its interaction with Nur77 in HCC cells. Although Nur77-mediated apoptosis continues to attract attention of researchers, how it is regulated in cancer cells remains unknown. In this proposal, I plan to investigate the effect of ALC1 on Nur77-mediated apoptosis, especially the effect on the translocation of Nur77 from nucleus to mitochondrial membrane. The effect of ALC1 on Nur77-mediated apoptosis pathway, including the release of Cyt c and activation of apoptosis-associated proteins (cleaved Casp 9 and Casp 3), will be addressed. Objectives: 1) To study the effect of ALC1 on the nucleus-mitochondria translocation of Nur77. 2) To study the effect of ALC1 on Nur77-mediated apoptosis pathway. Key issue and problem: Amplification of 1q21 is one of the most frequent genetic changes in HCC. Recently, we isolated a candidate oncogene ALC1 within 1q21 region. Although the oncogenic function of ALC1 has been clearly demonstrated by our previous in vitro and in vivo studies, the molecular mechanism of ALC1 in HCC development remains unknown. Our recent work showed that ALC1 interacts with Nur77, suggesting that the oncogenic function of ALC1 may be associated with its anti-apoptotic ability through the inhibiting Nur77-mediated apoptosis pathway. To elucidate the molecular mechanism of ALC1 in HCC development, two key issues will be addressed in this study. The first key issue is whether ALC1 is able to block the nucleus-mitochondria translocation of Nur77 when ALC1 binds to Nur77? The second key issue is whether ALC1/Nur77 interaction will inhibit Nur77-mediated apoptosis pathway? References: 1. Parkin DM, Bray F, Ferlay J, Pisani P. Global cancer statistics, 2002. CA Cancer J Clin 2005;55:74-108. 2. Wong N, Lai P, Lee SW, Fan S, Pang E, Liew CT, Sheng Z, Lau JW, Johnson PJ. Assessment of genetic changes in hepatocellular carcinoma by comparative genomic hybridization analysis: relationship to disease stage, tumor size, and cirrhosis. Am J Pathol 1999;154:37-43. 3. Guan XY, Fang Y, Sham JS, Kwong DL, Zhang Y, Liang Q, Li H, Zhou H, Trent JM. Recurrent chromosome alterations in hepatocellular carcinoma detected by comparative genomic hybridization. Genes Chromosomes Cancer 2000;29:110-6. 4. Ma NF, Hu L, Fung JM, Xie D, Zheng BJ, Chen L, Tang DJ, Fu L, Wu Z, Chen M, Fang Y, Guan XY. (2008). Isolation and characterization of a novel oncogene, amplified in liver cancer 1, within a commonly amplified region at 1q21 in hepatocellular carcinoma. Hepatology 2008;47:503-10. 5. Moll UM, Marchenko N, Zhang XK. p53 and Nur77/TR3 - transcription factors that directly target mitochondria for cell death induction. Oncogene 2006;25:4725-43. 6. Li P, Nijhawan D, Budihardjo I, Srinivasula SM, Ahmad M, Alnemri ES, Wang X. Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell 1997;91:479-89. 7. Li H, Kolluri SK, Gu J, Dawson MI, Cao X, Hobbs PD, Lin B, Chen G, Lu J, Lin F, et al. Cytochrome c release and apoptosis induced by mitochondrial targeting of nuclear orphan receptor TR3. Science 2000;289, 1159-64.

Project Title:	Evaluation of an Oxygen Therapeutic (IKOR-2084) in the Enhancement of Chemotherapy and Radiotherapy in Rodent Cancer Models
Investigator(s):	Guan XY
Department:	Clinical Oncology
Source(s) of Funding:	Innovation and Technology Fund Internship Programme
Start Date:	03/2009

Abstract:

Project Title:	Evaluation of an Oxygen Therapeutic (IKOR-2084) in the Enhancement of Chemotherapy and Radiotherapy in Rodent Cancer Models
Investigator(s):	Guan XY
Department:	Clinical Oncology
Source(s) of Funding:	Innovation and Technology Fund Internship Programme
Start Date:	03/2009

Abstract:

Project Title:	AACR 100th Annual Meeting 2009 FGFR2-positive fibroblasts provide a suitable microenvironment for tumor development and progression in esophageal carcinoma
Investigator(s):	Guan XY
Department:	Clinical Oncology
Source(s) of Funding:	URC/CRCG - Conference Grants for Teaching Staff
Start Date:	04/2009

Abstract:

N/A

Project Title:	Characterization of metastatic role of CHD1L in hepatocellular carcinoma
Investigator(s):	Guan XY
Department:	Clinical Oncology
Source(s) of Funding:	Small Project Funding
Start Date:	10/2009

Abstract:

Research Background: Hepatocellular carcinoma (HCC) is ranked as the fifth most frequent cancer in the world and affects one million people annually [1]. The prognosis of HCC is very poor, and the worldwide 5-year survival rate worldwide is less than 5%, mainly because of a high potential for vascular invasion, metastasis, and recurrence even after surgical resection [2]. Our previous study showed that amplification of 1q was one of the most frequent genetic alterations in primary hepatocellular carcinoma (HCC) [3]. Using a hybrid-selection strategy, we have isolated a candidate oncogene named chromodomain helicase DNA binding protein 1-like gene (CHD1L) from the 1q21 amplicon [4]. Our preliminary studies have indicated that the amplification and overexpression of CHD1L was detected in over 50% of HCC cases. In addition, CHD1L-transfected cells possess a strong oncogenic ability, including an increase in the colony formation capacity in soft agar and an increase in the tumorigenicity in nude mice, which could be effectively suppressed by using siRNA against CHD1L. Recently, we found that one of the oncogenic functions of CHD1L was associated with its apoptosis-inhibiting effect [5]. CHD1L belongs to SNF2-like family, which contains an SNF2_N domain and a helicase superfamily domain, suggesting that CHD1L may be able to stabilize protein-DNA interactions and play important roles in transcriptional regulation. In order to identify CHD1L-regulated genes, chromatin immunoprecipitation combined with subsequent cloning (ChIP-cloning) was applied to isolate DNA loci bound by CHD1L. Eighteen CHD1L-bound DNA sequences (all contain a specific CHD1L binding motif) and candidate CHD1L-regulated genes were isolated. One candidate CHD1L-regulated gene, RhoGEF9, was selected for further study because RhoGEF9 is a recently described regulator of the Rho GTPase-mediated signaling cascade. One of the important molecular mechanisms in cancer metastasis is the activation of the Rho family of small GTPases, which in turn leads to the rearrangement of the actin cytoskeleton and modulates cadherin-dependent cell-cell contacts. Therefore, characterization of the effect of CHD1L on HCC metastasis will greatly facilitate our understanding of the molecular mechanism of tumor metastasis. Objectives: The proposed study aims to elucidate the molecular mechanisms by which CHD1L manipulates the Rho GTPase signaling networks via the up-regulation of RhoGEF9 expression. Follows are two specific objectives: 1) To validate the up-regulatory effect of CHD1L on RhoGEF9 expression 2) To characterize the role of CHD1L/RhoGEF9 signaling in HCC metastasis Key issues and problems: Although the oncogenic function of CHD1L has been clearly demonstrated by our previous in vitro and in vivo studies, the molecular mechanism of CHD1L in HCC metastasis remains unknown. Our recent work has indicated that CHD1L can specifically bind to the SNF2-binding sequence and regulate the expression of the corresponding target gene. In this proposal, I select one target gene, RhoGEF9, for further study since Rho GTPase signaling networks have been associated with the development and progression of cancer by increasing cell proliferation and mobility. I hypothesize that the oncogenic function of CHD1L is associated with its role in manipulating the Rho GTPase signaling pathway via the transcriptional regulation of RhoGEF9 expression. The first key issue that will be addressed in this proposal is whether or not the expression of RhoGEF9 is regulated by CHD1L, and if this regulation is observed, I propose to determine the mechanism of regulation. Our preliminary data has indicated that the ectopic expression of CHD1L in both stable and transient CHD1L-transfectants is capable of up-regulating RhoGEF9 expression. Since the CHD1L-binding sequence is within the promoter region of RhoGEF9, it is likely that CHD1L is able to directly up-regulate RhoGEF9 expression as a transcription factor. In this proposal, the electrophoretic mobility shift assay will be used to confirm that CHD1L can specifically bind to the promoter region of RhoGEF9. The promoter region of RhoGEF9 will be also cloned into a vector containing the luciferase reporter gene, whereby luciferase activity will be evaluated to determine whether or not RhoGEF9 expression is regulated by CHD1L. The second key issue in this proposal is to determine whether or not the oncogenic function of CHD1L is facilitated through the activation of the Rho GTPase signaling pathway, and to determine the molecular mechanism for this process. Although the oncogenic role of CHD1L has been demonstrated in our previous studies, the molecular mechanism remains unclear. Since CHD1L can up-regulate the expression of RhoGEF9, it is reasonable to propose that the oncogenic function of CHD1L is facilitated through the activation of the Rho GTPases signaling pathway. The Rho GTPase signaling pathway has been associated with cell proliferation, cell invasion and migration. In this proposal, functional assays will be used to investigate the effect of CHD1L on cell growth, proliferation, invasion, and migration. In addition, in order to study the molecular mechanism, I will determine which downstream member of the Rho GTPases (Rho, Rac or Cdc42) is involved in the CHD1L-mediated pathway. I also propose to investigate whether or not the epithelial-mesenchymal transition (EMT) is involved in CHD1L-induced cell invasion and migration. References: [1] Thorgeirsson SS and Grisham JW. Molecular pathogenesis of human hepatocellular carcinoma. Nat Genet 2002;31:339-46. [2] Poon RT, Fan ST, Wong J. Risk factors, prevention, and management of postoperative recurrence after resection of hepatocellular carcinoma. Ann Surg 2000;232:10-24. [3] Guan XY, Fang Y, Sham JS, Kwong DL, Zhang Y, Liang Q, Li H, Zhou H, Trent JM. Recurrent chromosome alterations in hepatocellular carcinoma detected by comparative genomic hybridization. Genes Chromosomes Cancer 2000;29:110-6. [4] Ma NF, Hu L, Fung JM, Xie D, Zheng BJ, Chen L, Tang DJ, Fu L, Wu Z, Chen M, Fang Y, Guan XY. Isolation and characterization of a novel oncogene, amplified in liver cancer 1, within a commonly amplified region at 1q21 in hepatocellular carcinoma. Hepatology 2008;47:503-10. [5] Chen L, Hu L, Chan TH, Tsao GSW, Xie D, Huo KK, Fu L, Zheng BJ, Guan XY. Chromodomain helicase/adenosine triphosphatase DNA binding protein 1-like (CHD1L) suppresses the nucleus-to-mitochondria translocation of Nur77 to sustain hepatocellular carcinoma cell survival. Hepatology 2009;50:122-9.

List of Research Outputs

Chen L., Chan H.M., Man K. and Guan X.Y., Identification of the proteins Lsm2 and Apolipoprotein A-1 associated with liver regeneration by proteomic screeing (Abstract), The 16th International Liver Transplantation Society Congress, 16-19 June 2010, Hong Kong. Liver Transplantation. 2010, 16(6): s221.

He L.R., Liu M.Z., Li B.K., Rao H.L., Liao Y.J., Zhang L.J., Guan X.Y., Zeng Y.X. and Xie D., Clusterin as a predictor for chemoradiotherapy sensitivity and patient survival in esophageal squamous cell carcinoma., Cancer Science. 2009, 100: 2354-60.

He L.R., Liu M.Z., Li B.K., Rao H.L., Liao Y.J., Guan X.Y., Zeng Y.X. and Xie D., Prognostic impact of H3K27me3 expression on locoregional progression after chemoradiotherapy in esophageal squamous cell carcinoma., BMC Cancer. 2009, 9: 461.

Tang D., Dong S., Ma N.F., Xie D., Chen L., Fu L., Lau S.H., Li Y., Li Y. and Guan X.Y., Overexpression of eukaryotic initiation factor 5A2 enhances cell motility and promotes tumor metastasis in hepatocellular carcinoma, Hepatology. 2010, 51: 1255-63.

Wang Y., Liu Y.H., Mai S.J., He L.J., Laio Y.J., Deng H.X., Guan X.Y., Zeng Y.X., Kung H.F. and Xie D., Evaluation of serum clusterin as a surveillance tool for human hepatocellular carcinoma with hepatitis B virus related cirrhosis., J Gastroenterol Hepatol . 2010, 25: 1123-1128.

Wu G.Q., Xie D., Yang G.F., Liao Y.J., Mai S.J., Deng H.X., Sze J., Guan X.Y., Zeng Y.X., Lin M.C. and Kung H.F., Cell cycle-related kinase supports ovarian carcinoma cell proliferation via regulation of cyclin D1 and is a predictor of outcome in patients with ovarian carcinoma., International Journal of Cancer. 2009, 125: 2631-42.

Researcher : Guo S

List of Research Outputs

Guo S., Zhou J., Gao B., Hu J., Wang H., Meng J., Zhao X., Ma G., Lin C., Xiao Y., Tang W., Zhu X., Cheah K.S.E., Feng G., Chan D. and He L., Missense mutations in IHH impair Indian Hedgehog signaling in C3H10T1/2 cells: Implications for brachydactyly type A1, and new targets for Hedgehog signaling, Cell Mol Biol Lett. 2009, 15(1): 153-76.

Researcher : Ho BS

List of Research Outputs

Ho B.S., Cheng C.C.W., Leung K.K.H. and Cheah K.S.E., The role of Sox2 and Sox8 in mouse hair development, 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium, CUHK, Hong Kong, 15 May,. 2010.

Researcher : Ho DWH

List of Research Outputs

Cheung K.M.C., Samartzis D., Karppinen J., Mok P.S., Ho D.W.H., Fong D.Y.T. and Luk K.D.K., “Skipped” level disc degeneration of the lumbar spine, a MRI study of 1989 individuals, International Society for the Study of the Lumbar Spine Annual Meeting, Auckland, New Zealand, April 13-17, 2010.

Kao Y.P.P., Chan D., Cheah K.S.E., Cheung K.M.C., Ho D.W.H., Karppinen J., Leong J.C.Y., Yip S.P., Song Y. and Sham P.C., Genome-wide Association Study of Degenerative Disc Disease (DDD), 14th Research Postgraduate Symposium, December 2 & 3, 2009, The University of Hong Kong. 2009.

Researcher : Hu J

List of Research Outputs

Guo S., Zhou J., Gao B., Hu J., Wang H., Meng J., Zhao X., Ma G., Lin C., Xiao Y., Tang W., Zhu X., Cheah K.S.E., Feng G., Chan D. and He L., Missense mutations in IHH impair Indian Hedgehog signaling in C3H10T1/2 cells: Implications for brachydactyly type A1, and new targets for Hedgehog signaling, Cell Mol Biol Lett. 2009, 15(1): 153-76.

Researcher : Huang J

Project Title:	RNA-DNA hybrid oligonucleotide mediated site-specific repair and gene therapy
Investigator(s):	Huang J
Department:	Biochemistry
Source(s) of Funding:	Matching Fund for Hi-Tech Research and Development Program of China (863 Projects)
Start Date:	01/2003

Abstract:

To establish the reporter system for RDO and SSO mediated mutagenesis in mouse ES cells; to investigate the methods to improve the mutagenesis efficiency of RDO- and SSO-mediated mutagenesis; to study the mechanisms underlying SSO-mediated mutagenesis.

Project Title:	Towards a visualized proteome of Escherichia coli
Investigator(s):	Huang J, Danchin A, Smith DK
Department:	Biochemistry
Source(s) of Funding:	General Research Fund (GRF)
Start Date:	10/2006
Completion Date:	09/2009

Abstract:

The aim are (1) resources development: generate EGFP-tagged E. coli strain library for systematic analysis of E. coli proteome; (2) functional analysis: characterization of the subcellular localization and dynamics of the E. coli proteome; (3) bioinformatics studies: cataloging and annotating E. coli protein localization information that allows interactive queries, followed by proteomic studies to define the relationships between subcellular sites and protein functions.

Project Title:	Development of bacteria-mediated oral vaccine against highly pathogenic avian influenza viruses for veterinary use
Investigator(s):	Huang J, Yuen KY, Zheng B
Department:	Biochemistry
Source(s) of Funding:	Research Fund for the Control of Infectious Diseases - Full Grants
Start Date:	11/2007

Abstract:

To establish an integrated recombineering platform for vaccine development and production; to develop a novel bacteria-mediated oral vaccine against avian influenza viruses for veterinary use.

Project Title:	Toward the treatment of breast cancer by targeting breast tumor initiating cells with microRNAs
Investigator(s):	Huang J
Department:	Biochemistry
Source(s) of Funding:	Travel Grants for NSFC/RGC JRS
Start Date:	12/2007

Abstract:

Travel grants for NSFC/RGC JRS.

Project Title:	Development of an undergraduate teaching curriculum in Synthetic Biology
Investigator(s):	Huang J, Cheah KSE, Zhang FC, Chen G, Shum DKY, Wu EX
Department:	Biochemistry
Source(s) of Funding:	Run Run Shaw Research and Teaching Endowment Fund - Teaching Grants
Start Date:	07/2008

Abstract:

What is synthetic biology? Synthetic biology studies aim to design and fabricate of artificial biological parts, devices, systems, and organisms for engineering applications. It is an engineering application of our knowledge of biological science, to benefit human race and for the exploration of scientific problems. It relies on design and fabrication of biological components and systems that do not already exist in the natural world and on the re-design and utilization of existing biological systems. Why synthetic biology is different from current biological science and conventional genetic engineering? Science emphasizes on “unfiltered observations, analyzing data with an open mind and conducting value-neutral experiments” (1). A scientific conclusion does not need to meet pre-determined idea. To understand a biological phenomenon such as “a biological clock”, scientific experiments can be performed to form a theory of how “a biological clock” might work. It does not matter whether the theory reflects the actual way how “a biological clock” works as long as it can accommodate all the observations. On the other hand, engineering sets a defined goal such as ‘build-a biological clock’. To achieve this goal, engineers not only have to have a complete understanding of all the biological parts and theories of clock, but they also have to be able to build and assemble these parts into a functional one. When a problem is encountered, it cannot just be ignored by engineers while problems are often set aside in science. Engineers must then seek a solution perhaps by entering scientific areas that are normally ignored by either observation or analysis. For this reason, synthesis biology will promote the development of biological sciences. Conventional genetic engineering usually changes one or two genes of an organism. Synthetic Biology distinguishes itself from conventional genetic engineering by emphasis on developing standardized technologies and standardized parts to rewire the genetic circuits of an organism. It is a true engineering discipline. Objectives: We aim to develop a curriculum in Synthetic Biology consists of lectures, case studies, and practical. Specifically, we will develop the following: • Lectures: introduction of the basic concepts of synthetic biology • Case studies: analysis of successful design and fabrication of biological devices and circuits; analysis of the wining projects in the previous international Genetically Engineered Machines competition. • Practical: design and build engineered biological systems by HKU students; participate in the international Genetically Engineered Machines competition (iGEM).

Project Title:	Pattern formation via cell mobility
Investigator(s):	Huang J
Department:	Biochemistry
Source(s) of Funding:	Small Project Funding
Start Date:	12/2008
Completion Date:	11/2009

Abstract:

Pattern formation is one of the most common yet fascinating biological phenomena happening in living organisms. For centuries, biologists, physicists and mathematicians have struggled to understand its nature. How do highly ordered patterns arise from a few living cells? How can our hands, our eyes, our bones form their shape with such an extremely low mistake rate? This question is fascinating but crucial. In this project, we will take an engineering approach to design and generate artificial patterns using living cells. There are many possible way to form highly complicated patterns. Controlled cell movement is one of the many possible mechanisms. Here, we will focus on pattern formation by using controlled and coordinatied cell movement. The aim of our project is to generate a simple pattern, a set of concentric circles based on a strain of E. coli that we are going to create.

Project Title:	Toward a bioshield against influenza virus infection by commensal bacteria secreting antiviral peptide
Investigator(s):	Huang J, Yuen KY, Zheng B
Department:	Biochemistry
Source(s) of Funding:	Research Fund for the Control of Infectious Diseases - Full Grants
Start Date:	12/2008

Abstract:

(1) Genetically engineer a naturally occurring bacteria to provide protection against H5N1 by secreting antiviral peptide that interfere with viral attachment; (2) Introduce such genetically modified organisms to colonize in animals as a long-lasting biosheidl; (3) Test the prevention efficacy against H5N1 in a mouse model

Project Title:	Toward the treatment of breast cancer by targeting breast tumor initiating cells with microRNAs
Investigator(s):	Huang J
Department:	Biochemistry
Source(s) of Funding:	NSFC/RGC Joint Research Scheme
Start Date:	04/2009

Abstract:

(1) Characterization of miRNAs that are differentially expressed in BT-IC and SK-3rd and SKBR3; (2) Development of antibody-protamine fusion proteins as miRNA targeting vector to BT-IC; (3) Engineer a conditional anaerobic bacterium as miRNA targeting vector to BT-IC (i) improve the safety and tumor-targeting ability of Salmonella; (ii) increase the bacteria-BT-IC affinity (iii) delivery of miRNA into BT-IC by bacteria-based vectors.

Project Title:	Development of a Pan-organism Riboregulator Device
Investigator(s):	Huang J, Jin Y
Department:	Biochemistry
Source(s) of Funding:	Innovation and Technology Support Programme (Tier 3)
Start Date:	11/2009

Abstract:

There are three objectives of this project. The first objective is to design and assemble a riboregulator device specific for enhanced green fluorescence protein; the second objective is to test the portability of the riboregulator in various organisms: bacteria and yeast, and mammalian cells in the future; the third objective is to test the extensibility of the riboregulators which are rationally designed to control the expression of other gene targets.

Project Title:	Development of a Pan-organism Riboregulator Device
Investigator(s):	Huang J
Department:	Biochemistry
Source(s) of Funding:	Innovation and Technology Fund Internship Programme
Start Date:	12/2009

Abstract:

Project Title:	Study of kinesin-1-dependent transport of α-sarcomeric actin for myofibril assembly
Investigator(s):	Huang J
Department:	Biochemistry
Source(s) of Funding:	Seed Funding Programme for Basic Research
Start Date:	04/2010

Abstract:

Skeletal muscle has a remarkably ordered cytoskeleton, exhibiting repeated cross-striations mainly composed of actin and myosin molecules. The relative sliding of actin and myosin causes muscle contraction (1). Whilst significant progress has been made in understanding muscle structure and contraction, the molecular mechanisms for step-by-step construction of the muscle structure are less clear. To build the complex structure of myofibers, somite cells first undergo differentiation to form myotome cells that are committed to become myoblasts by expression of proteins such as myogenic regulatory factors. Later, myoblasts exit the proliferating cycle and change from a polygonal to a bipolar shape, then align in the same direction and fuse with each other. Newly expressed muscle specific proteins are then transported to their appropriate sites for myofibril assembly. We are particularly interested in the transportation of the muscle specific proteins for myofibril assembly. Microtubules and actin filaments have been shown to be active in generating the spatial organization in differentiating muscle cells (2-5). However, which motor molecules are responsible for the delivery of myofibril components such as myosin and actin to the sites of assembly are previously unknown. Our preliminary works on kinesin-1 suggest that it transports a subset of the myofibril components in differentiating myoblasts. The data for our findings are summarized below. Kif5b conditional knockout in muscles We depleted kineisn-1 in muscle by using conditional Kif5b (heavy chain of kinesin-1) knockout mice. While the wild type newborn mice quickly started to breathe, the mutant mice died within minutes with noninflated lungs. One prominent phenotype consistently observed was severe muscle dystrophy. We first examined the structure of muscles in newborn mice under light microscope. We found that while control muscle cells were arranged in parallel with visible striations, mutant cells were disorganized with different sizes, and no striation was observed (Fig. 1a&c). Muscles were then examined at the ultrastructural level. Rudimentarily assembled or non-assembled myofibrils were consistently observed in mutant muscle tissues (Fig. 1b). We stained myosin II (MyoII) and actin as two major components in thick and thin filaments respectively to examine myofibril assembly. Wild type muscle fibers exhibited regular striated pattern, but in mutant muscle fibers, only a small amount of MyoII and actin was observed in poorly assembled structures adjacent to the plasma membrane, while the remaining MyoII and actin was distributed irregularly within the cell (Fig. 1c). Taken together, these data indicate an important role of Kif5b in myogenesis. An in vitro muscle differentiation system to study kinesin-1 function Myogenesis is a multi-step event which includes differentiation stage-specific gene regulation, cell shape change, cell fusion, and assembly of myofibrils. Since Kif5b could involve in many aspects of myogensis, we examined the mutant cells for defects in proliferation and differentiation. We first established an in vitro differentiation system by using primary culture satellite cells. Satellite cells are adult muscle stem cells residing underneath the basal lamina that surrounds muscle fibers and adjacent to the plasma membrane of mature muscle fibers. Upon activation such as injury, they enter a proliferating state and undergo myoblast differentiation and fusion to regenerate muscle fibers (reviewed in (6,7)). This in vitro differentiation system has been used to study the differentiation process of muscles (8). We extracted satellite cells from wild type and mutant mice. We then compared the wild type and mutant cells for their ability for proliferation, cell fusion and expression of stage-specific differentiation markers for muscle formation. We found that the mutant satellite cells are capable of proliferation, expression of muscle specific genes, and cell fusion. However, the mutant cells are defective in myofibril assembly (data not shown). Kif5b is responsible for α-sarcomeric actin localization Considering the defects in assembly of myofibrils, and the transportation function of Kif5b as a molecular motor, we suspected that Kif5b may be responsible for transportation of proteins involved myofilament assembly. We therefore checked the localization of a panel of marker proteins in differentiating myoblasts. The distribution of most proteins examined, such as MyoII, was not affected in mutant cells, showing that Kif5b may be not directly involved in their transportation and localization. In contrast, the localization of myofilament component α-sarcomeric actin (Fig. 2) was severely affected in mutant cells. Instead of being transported to the terminus of the myotube in wild type cells where the assembly of myofilaments initiates (9), α-sarcomeric actin aggregated around the cell nuclei in mutant cells. To prove that Kif5b is responsible for the localization of α-sarcomeric actin, we did a rescue experiment by transfecting Kif5b expression vectors into the mutant cells. While expression of a Kif5b without the motor domain did not change protein localization, expression of full-length Kif5b dramatically stimulated dispersion of these protein into the cell terminus (Fig. 2). These data indicate that Kif5b functions in the transportation of α-sarcomeric actin to myofilament assembly sites, and this function depends on the kinesin motor domain. Interaction between kinesin-1 and α-sarcomeric actin in muscle To verify α-sarcomeric actin as a cargo of kinesin-1, we performed coimmunoprecipitation followed by Western blot (Fig. 3a). The result showed that α-sarcomeric actin can be coimmunoprecipitated with Kif5b. The interaction between α-sarcomeric actin and Kif5b was further supported by the co-localization of these proteins in skeletal muscles. While distributed uniformly in the cell body in in vitro cultured myoblasts (Fig. 3), Kif5b appeared striated pattern around the Z-disks (visualized by coimmunostaining with MyoII and other markers) in mature mice muscle (Fig. 3b). However, key issues remain: the exact mechanisms how kinesin-1 delivers myofibril components is unknown. Kinesin-1 can deliver myofibril components by directly binding to the myofibril components or through mediator proteins. We propose to carry out a mechanistic study on kinesin-1 mediated transport of myofibril components. The objective of this proposal is to reveal the mechanisms of kinesin-1-dependent myofibril assembly. Due to the small scale of this grant, we only aim to identify the subunits and domains of kinesin-1 responsible for the delivery of myofibril components in differentiating myoblast by using kinesin-1 mutant constructs and siRNA technology.

Project Title:	14th HUMAN GENOME MEETING 2010 Genomic scale labeling of persistent genes
Investigator(s):	Huang J
Department:	Biochemistry
Source(s) of Funding:	URC/CRCG - Conference Grants for Teaching Staff
Start Date:	05/2010
Completion Date:	05/2010

Abstract:

N/A

Project Title:	Programming Tumor-targeting Bacteria by Combinatorial Control
Investigator(s):	Huang J
Department:	Biochemistry
Source(s) of Funding:	Seed Funding Programme for Applied Research
Start Date:	06/2010

Abstract:

Introduction Cancer is a leading cause of death despite many years of research. Metastasis, relapse, and resistance to chemo- and radiotherapy are the main reason for lethality. This has prompted the development of many new approaches for the treatment of cancer, including the delivery of anti-cancer genes to tumors using viral and non-viral vectors. However, these approaches have inadequate specificity for tumors. The vectors used usually cannot replicate and are often short-lived. Even when they are in tumors, these vectors are poorly distributed throughout the tumor mass. Therefore, novel system is required to target both primary and metastatic tumors. One interesting method is to use bacteria as cancer therapeutic agent. The first reported treatment of tumor by bacteria was about 150 years ago. In 1868, the German physician W. Busch found that one of his sarcoma patient got the infection of “erysipelas” (Streptococcus pyrogenes), and surprisingly the primary tumor size was shrunk by a half within a week. Since bacteria can sense their environment, distinguish between cell types, and deliver proteins and RNAs to eukaryotic cells, current advances in synthetic biology technology have brought bacteria-tumor therapy back in focus. We hypothesize that bacteria can be programmed to kill cancer cells with high efficacy and specificity. We have already obtained encouraging data in vitro and in vivo using genetically engineered strains of Salmonella according to our preliminary designs. Preliminary results (1) Bacteria can be programmed to preferentially accumulate in tumors. We set out to program tumor-specific accumulation of bacteria using artificial genetic circuits. Our strategy is to control the expression of a gene essential (asd) for bacterial growth by a generic circuit which specifically turns this gene on inside the tumor but not in normal tissues (Figure 1a). Hypoxic condition is chosen as the cue for tumor-specific gene expression because in addition to its widespread existence in tumors, and its known role in resistance to both radiation and drug treatment, it has been proposed as niches for the maintenance of cancer stem cell (1). The responsiveness of the hypoxia-controlled circuit to O2 was tested by culturing the programmed Salmonella (termed SL7207-37) under anaerobic and aerobic conditions in a hypoxia chamber. The results demonstrate that our construct allowed bacterial growth only under low O2 levels (Figure 1b). By using tumor-bearing nude mice, we found that the accumulation of SL7207-37 was about 1,000–fold less than its cogenic parent SL7207 in most normal organs examined while remaining similar in tumor (Figure 1c). The enhanced preferential accumulation of engineered bacteria in tumor coincided with reduced mortality rate (Figure 2b). Together, these results demonstrate that (i) hypoxia control can indeed be used for preferential gene expression in tumors, and (ii) a high degree of preferential bacteria accumulation can be achieved inside solid tumors for an extended period of time. (2) Tumor repression effect. We further found that the engineered bacteria not only allow the survival of the animals but also repress tumor growth of both breast and liver (Figure 2c) cancers. Figure 2a shows that a single dose (5×10e7 cfu) bacterial treatment can already retard tumor growth for an extended period of time, and interestingly, the synthetic strain SL7207-37 has a better tumor-repression effect than its cogenic parent SL7207 even though their level in tumor cells are similar (Figure 1c). Objectives However, the bacterial treatment did not shrink the tumor size down to zero. In this project, we will design and construct bacterial strains to improve the tumor-targeting and tumor-killing specificity by using combinatorial controls, and evaluate their efficacies in vitro and in vivo using animal model.

List of Research Outputs

Chan C.M., Lau S.K.P., Woo P.C.Y., Tse H., Zheng B., Chen L., Huang J. and Yuen K.Y., Identification Of Major Histocompatibility Complex Class I C Molecule As An Attachment Factor That Facilitates Coronavirus Hku1 Spike-mediated Infection, Journal of Virology. 2009, 83: 1026-1035.

Chen D., Zeng Y., Zhou J., Yang L., Jiang S., Huang J., Lu L. and Zheng B., Association of candidate susceptible loci with chronic infection with hepatitis B virus in a Chinese population. , J Med Virol.. 2010, 82(3):: 371-378.

Chen M., Huang J., Hu L., Zheng B., Chen L., Tsang S.L. and Guan X.Y., Transgenic CHD1L expression in mouse induces spontaneous tumors, PLoS One. 2009, 4: e6727.

Cui J., Wang Z., Cheng Q.N., Lin R., Zhang X., Leung P.S., Copeland N.G., Jenkins N.A., Yao K.M. and Huang J., Targeted Inactivation of KIF5B in Pancreatic Leads to Insulin Secretory Deficiency and Increased b-Cells Mass in vivo, 14th Research Postgraduate Symposium, December 2 & 3, 2009, The University of Hong Kong. 2009.

Huang J., Sun H., Tanner J.A. and Watt R.M., Determine the functions of the putative metal-binding domain of SARS-CoV helicase, Hong Kong Medical Journal. 2009, 15(5) Supplement: 6:15-16.

Huang J., Gold Award, International Genetically Engineered Machine Competition 2009, MIT. 2009.

Huang J., Invited Speech: Can bacteria be programmed to cure cancer? , Institut CURIE, Recherche, CNRS UMR2027, Bât 110, Centre Universitaire, F-91405 ORSAY, France, May 17, 2010 . 2010.

Huang J., Invited Speech:, 新颖生物图形的人工编码、合成及数学模型的建立, Academy of Mathematics and Systems Science, Chinese Academy of Science, Beijing, China, May 24, 2010 . 2010.

Huang J., Invited speech: Creation of biological patterns controlled by synthetic genetic circuits, Peking Union Medical College and the Chinese Academy of Medical Sciences, Beijing, China, June 11, 2010 . 2010.

Huang J., Invited speech: Creation of biological patterns controlled by synthetic genetic circuits, The National Institute of Biological Sciences, Beijing, Beijing, China, June 10, 2010 . 2010.

Huang J., Symplectic Biology, Fondation Fourmentin-Guilbert, 2010.

Huang J., 编码细胞运动特性来形成新颖的图形, College of Life Science, Jilin University, Changchun, Jilin, China. 2009.

Huang J., 编码细胞运动特性来形成新颖的图形, State Key Laboratory of Pathogen and Biosecurity, Department of Pathogenic Molecular Biology, Institute of Microbiology and Epidemiology, The Academy of Military Medical Science, Beijing, China . 2009.

Jin Y., Watt R.M., Danchin A. and Huang J., Use of a riboswitch-controlled conditional hypomorphic mutation to uncover a role for the essential csrA gene in bacterial autoaggregation, Journal of Biological Chemistry. 2009, 284: 28738-28745.

Kao P.Y.P., Yang D., Lau L.S., Tsui H.W., Hu L., Dai J., Chan M.P., Chan C.M., Wang P., Zheng B., Sun J., Huang J., Madar J., Chen G., Chen H., Guan Y. and Yuen K.Y., Identification of Influenza a Nucleoprotein as an Antiviral Target, Nature Biotechnology. 2010, 28: 600-608.

Lin R., Lau C.F., Wang J., Ni Y., Fung M.L. and Huang J., Reduced neuronal nitric oxide production in KIF5b-deficiency mice, 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium, CUHK, Hong Kong, 15 May,. 2010.

Liu C., Wang Z., Huen M.S.Y., Lu L., Liu D. and Huang J., Cell death caused by single-stranded oligodeoxynucleotides mediated targeted genomic sequence modification, Oligonucleotides. 2009, 19(3): 281-286.

Liu C., Fu X.F., Ren X.J., Chen G.H., Tang L.H., Hwa T.T. and Huang J., Formation of Novel Pattern by Programming Cell Motility, 14th Research Postgraduate Symposium, December 2 & 3, 2009, The University of Hong Kong. 2009.

Liu C., Fu X., Chen G., Tang L.H., Hwa T. and Huang J., Formation of novel pattern by programming cell motility, 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium, CUHK, Hong Kong, 15 May,. 2010.

Lu S., Lu L., Guan X.Y. and Huang J., The Phenotype Study of Pdss2 Conditional Knockout Mouse, 14th Research Postgraduate Symposium, December 2 & 3, 2009, The University of Hong Kong. 2009.

Miao Y., Danchin A.L.M. and Huang J., Visualizing the Proteome Escherichia coli by Recombineering Technology, 14th Research Postgraduate Symposium, December 2 & 3, 2009, The University of Hong Kong. 2009.

Qi S., Sun H. and Huang J., Expression and Purification of Mutated hpn Protein, and Studies of Function of Cystein Residues in hpn, 14th Research Postgraduate Symposium, December 2 & 3, 2009, The University of Hong Kong. 2009.

Qi S., Sun H. and Huang J., Expression and purification of mutated hpn protein and studies of cysteine residues in hpn, 14th International Conference on Biological Inorganic Chemistry, Nagoya, Japan, July 25-30, 2009 (published in J. Biol Inorg Chem. 2009, 14, Suppl1, P606). 2009.

Shi L., Yu B., Yang M. and Huang J., Engineering Tumor-killing Salmonella against Breast Cancer, 14th Research Postgraduate Symposium, December 2 & 3, 2009, The University of Hong Kong. 2009.

Xie L...X..., Durairajan S...S...K..., Lu J...H..., Liu C., Liu C., Kum W...F..., Wang Y., Koo I., Wu W...K..., Han D., Lao F., Huang J. and Li M., The effect of salvianolic acid B combined with laminar shear stress on TNF--stimulated adhesion molecule expression in human aortic endothelial cells, Clinical Hemorheology and Microcirculation. 2010, 44: 245–258.

Zhou J., Huang J., Poon K.M., Chen D., Chan C.S., Ng F., Guan X.Y., Watt R.M., Lu L., Yuen K.Y. and Zheng B., Functional dissection of an IFN-alfa/belta receptor 1 promoter variant that confers higher risk to chronic hepatitis B virus infection. , J Hepat.. 2009, 51(2):: 322-332.

Researcher : Hui CC

List of Research Outputs

Wang X., Wong E.Y.M., Hui C.C. and Sham M.H., Hoxb1 Cooperates with Gli to Mediate Sonic Hedgehog Signaling in Hindbrain Neurogenesis , 14th Research Postgraduate Symposium, December 2 & 3, 2009, The University of Hong Kong. 2009.

Wang X., Wong E.Y.M., Hui C.C. and Sham M.H., Hoxb1 cooperates with GL1 to mediate sonic hedgehog signaling in hindbrain neural progenitor cell fate determination, International Society for Stem Cell Research, 8th Annual Meeting, Moscone West, San Francisco, CA, USA, June 16-19, 2010.

Wang X., Wong E.Y.M., Hui C.C. and Sham M.H., Hoxb1 cooperates with Gli to activate NKx2.2 expression in the neurogenesis of mammalian hind brain rhombomere 4, 43rd Annual Meeting for the Japanese Society of Developmental Biologists, Jointly Sponsored by the Asia-Pacific Developmental Biology Network, Kyoto, Japan, 20-23 June. 2010.

Wang X., Wong E.Y.M., Hui C.C. and Sham M.H., Hoxb1 cooperates with Gli to activate Nkx2.2 expression in the neurogenesis of mammalian hindbrain rhombomere 4, 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium, CUHK, Hong Kong, 15 May,. 2010.

Wang X., Wong Y.M.E., Hui C.C. and Sham M.H., Hoxb1 cooperates with Gli to active Nkx2.2 expression in the neurogenesis of mammalian hind brain rhombomere 4., 43rd Annual Meeting for the Japanese Society of Developmental Biologists Kyoto, Japan, 20 June 2010 . 2010.

Researcher : Ip CH

List of Research Outputs

Ip C.H., Chan C.H. and Shum D.K.Y., Serglycin in the HL-60 degranulation product retains its sulfated chondroitin/dermatan moieties and affinity for neutrophil elastase, 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium, CUHK, Hong Kong, 15 May,. 2010.

Researcher : Jia Y

List of Research Outputs

Jia Y., Song Y., Yiu S.M. and Smith D.K., Bioinformatics Study of the Lineage and Tissue Specificity of Gene Expression, 14th Research Postgraduate Symposium, December 2 & 3, 2009, The University of Hong Kong. 2009.

Jia Y., Bioinformatics study of the Lineage and Tissue Specificity of Genes and Gene Expression , M.Phil Thesis, 2010.

Researcher : Jia Y

List of Research Outputs

Jia Y., Bioinformatics study of the Lineage and Tissue Specificity of Genes and Gene Expression , M.Phil Thesis, 2010.

Researcher : Jin D

Project Title:	Mitotic checkpoint and genomic stability in ovarian cancer
Investigator(s):	Jin D
Department:	Biochemistry
Source(s) of Funding:	National Institutes of Health, US Department of Health and Human Services - General Award
Start Date:	09/2002

Abstract:

To investigate the molecular basis of mitotic checkpoint in mammalian cells nad its relevance to genomic instability in ovarian cancer.

Project Title:	Roles for p21-activated protein kinase 3 in HTLV-I pathogenesis
Investigator(s):	Jin D, Ching YP
Department:	Biochemistry
Source(s) of Funding:	General Research Fund (GRF)
Start Date:	01/2006

Abstract:

To characterize the expression of Pak 3 in HTLV-I-transformed ATL cells; to study whether and how Tax induces Pak 3 expression and activity; to define the contributory roles of Pak 3 in Tax modulation of transcription and cell signaling; to assess the significance of Tax-Pak3 interaction in viral transformation and leukemogenesis.

Project Title:	Characterization of fusion oncoprotein FUS-CREB3L2 found in soft tissue sarcoma
Investigator(s):	Jin D, Sham MH
Department:	Biochemistry
Source(s) of Funding:	General Research Fund (GRF)
Start Date:	01/2007
Completion Date:	12/2009

Abstract:

To characterize the transforming activity of FUS-CREB3L2 in murine fibroblasts and mice; to determine the subcellular localization and transcriptional activity of FUS-CREB3L2; to identify the cellular targets of FUS-CREB3L2 using cDNA microarray; to create FUS-CREB3L2 transgenic mice for the study of this fusion oncoprotein.

Project Title:	Roles and regulation of yeast peroxiredoxins in antioxidant defense and in the maintenance of genome stability
Investigator(s):	Jin D
Department:	Biochemistry
Source(s) of Funding:	General Research Fund (GRF)
Start Date:	01/2008

Abstract:

To define the molecular basis of the interaction between Tsa1p and Apd1p; to characterize the biochemical and biological properties of Apd1p; to investigate the biological significance of Tsa1p-Apd1p interaction. Particularly, we will ask whether Apd1p serves as an electron donor for Tsa1p; to evaluate the sensitivity of peroxiredoxin-null cells to DNA-damaging agents and screen for suppressors of drug sensitivity and synthetic lethality; to assess oxidative DNA damage in peroxiredoxin-null cells and determine the competence of DNA checkpoint and repair machinery.

Project Title:	Characterization of a novel growth-suppressive isoform of TRBP in liver cancer
Investigator(s):	Jin D, Kok KH
Department:	Biochemistry
Source(s) of Funding:	Seed Funding Programme for Basic Research
Start Date:	05/2008
Completion Date:	04/2010

Abstract:

Project Objectives: 1) Characterization of TRBP-beta isoform in liver cancer 2) Tumor-suppressive properties of TRBP-beta 3) Functional characterization of TRBP-beta Major hypothesis: TRBP-beta, which lacks a critical double-stranded RNA-binding domain, counteracts the oncogenic activity of TRBP-alpha and functions as a tumor suppressor protein in liver cancer. Key issues and problems being addressed: 1) Underexpression of TRBP-beta in liver cancer cells: A full documentation of the expression pattern of TRBP-beta as well as its correlation with clinical and pathological features will provide a key evidence for the tumor suppressive role of this isoform. 2) Tumor suppressive activity of TRBP-beta: Demonstration of the growth suppressive properties of TRBP-beta as well as the verification of its dominant interfering activity on TRBP-alpha using different assays conducted in cultured cells will lend further support to our model. 3) Further characterization of TRBP-alpha: Comparison of the RNA-binding, PKR-regulating and RNAi-modulating activities of the two TRBP isoforms will shed additional light on the molecular mechanisms underlying the tumor suppressor function of TRBP-beta.

Project Title:	Outstanding Researcher Award 2007-2008
Investigator(s):	Jin D
Department:	Biochemistry
Source(s) of Funding:	Outstanding Researcher Award
Start Date:	10/2008

Abstract:

The Awards are intended to recognize, reward, and promote exceptional research accomplishments of academic and research staff.

Project Title:	Roles and regulation of MIP-T3 in centrosomal and ciliary function
Investigator(s):	Jin D, Ching YP, Kok KH
Department:	Biochemistry
Source(s) of Funding:	NSFC/RGC Joint Research Scheme
Start Date:	01/2009

Abstract:

(1) Interaction of MIP-T3 with centrosomal and ciliary coiled-coil proteins; (2) roles and regulation of MIP-T3 in centrosomal duplication and/or seperation; (3) Roles and regulation of MIP-T3 in primary cilium assembly and intraflagellar transport; (4) roles of MIP-T3 and related proteins in breast cancer development.

Project Title:	Posttranslational modifications of TORC transcriptional coactivators and implications in HTLV-1 pathogenesis
Investigator(s):	Jin D
Department:	Biochemistry
Source(s) of Funding:	General Research Fund (GRF)
Start Date:	01/2009

Abstract:

(1) Characterize MEKK1-mediated phosphorylation of TORC transcriptional coactivators: verify TORC phosphorylation in vitro and in vivo; determine phosphorylation sites; define molecular basis of MEKK1-induced TORC phosphorylation; characterize upstream modulators and downstream effectors; assess influence on TORC localization and activity; (2) Characterize MEKK1-mediated ubiquitination of TORC transcriptional coactivators: verify TORC ubiquitination in vitro and in vivo; determine ubiquitination sites; define molecular basis of MEKK1-induced TORC ubiquitination; assess influence on TORC localization and activity; (3) Investigate the implications of MEKK1-induced phosphorylation and ubiquitination of TORCs in HTLV-1 pathogenesis: document expression and activity profiles of TORCs and TORC regulators in HTLV-1-transformed cells; assess the influence of Tax on TORC phosphorylation and ubiquitination; investigate the mechanism of Tax-induced activation of TORCs; define the roles of TORC activation in cell proliferation and transformation.

Project Title:	Characterization of a novel cellular target of Epstein-Barr virus-encoded miRNA miR-BART3
Investigator(s):	Jin D
Department:	Biochemistry
Source(s) of Funding:	Seed Funding Programme for Basic Research
Start Date:	05/2009

Abstract:

Key issues and questions being addressed: Epstein-Barr virus (EBV) is the first human virus shown to be etiologically associated with cancer. Although EBV establishes a lifelong latent infection in more than 90% of the world's population without serious sequelae, a small fraction of latently infected individuals develop malignancies of lymphocytic and epithelial origin, such as Burkitt's lymphoma, Hodgkin's lymphoma, extranodal nasal natural killer/T cell lymphoma, nasopharyngeal carcinoma (NPC) and gastric cancer. NPC is a malignancy arising from the epithelial cells lining the nasopharynx. It is particularly prevalent in Hong Kong and adjacent areas of China. While the etiology of NPC is complex and multifactorial, it is thought that the infection of EBV in premalignant nasopharyngeal epithelial cells drives malignant transformation to full scale. Among over a hundred viral genes expressed during productive replication, only less than a dozen are expressed in latently infected epithelial cells. EBV is also the first human virus found to encode microRNAs (miRNAs). miRNAs are gene regulatory RNAs of ~22-nucleotides in length and they negatively regulate gene expression through specific mRNA cleavage or translational repression. Cellular miRNAs are critically involved in various biological processes such as development, apoptosis, immune response and cell proliferation. EBV encodes more than 20 miRNAs, which are organized within the viral genome in two clusters termed miR-BARTs and miR-BHRFs. It is noteworthy that miR-BARTs are among a small number of viral genes abundantly expressed in latently infected epithelial cells but at significantly lower level in B cells. Thus, characterization of these miRNAs may provide mechanistic insight into the establishment of latency and the development of epithelial cancer. The functions of most EBV miRNAs are poorly understood. While recent reports suggest that they serve important roles in regulating viral gene expression, they may also target cellular transcripts to facilitate viral persistence and oncogenesis. Indeed, potential cellular targets of EBV miRNAs have been predicted by in silico analysis. In addition, reports on experimental validation of these targets have just begun to appear in the literature. In this regard, we are taking a lead internationally in the identification and characterization of miR-BART5 targets. In a recent work, we have identified p53 upregulated modulator of apoptosis (PUMA) to be a cellular target of miR-BART5, an EBV miRNA abundantly expressed in NPC and EBV-associated gastric carcinoma cells. We have further demonstrated the modulation of PUMA expression by miR-BART5 and its consequent antiapoptotic function in EBV+ cells and NPC tissues. Our findings suggest that an EBV miRNA facilitates the establishment of latent infection in epithelial cells and the development of cancer by promoting host cell survival (Choy et al. J. Exp. Med. 2008 Oct 27; 205:2551-2560). Objectives: 1) We will verify the modulation of INTS6 expression by miR-BART3 and anti-miR-BART3 in EBV+ epithelial carcinoma cells such as C666-1 and AGS/BX1. 2) We will confirm the underexpression of INTS6 in EBV-associated epithelial cancer cells and primary tumor tissues. 3) We will establish the roles of miR-BART3 and INTS6 in regulating cellular growth, proliferation and transformation.

Project Title:	4th Intracellular Proteolysis Meeting on Ubiquitin-proteasome System, Dynamics and Targeting REV7-mediated polyubiquitination and degradation of human REV1
Investigator(s):	Jin D
Department:	Biochemistry
Source(s) of Funding:	URC/CRCG - Conference Grants for Teaching Staff
Start Date:	05/2009

Abstract:

N/A

Project Title:	Knockdown of miR-BART5 in EBV-positive epithelial cells by RNA decoys delivered through novel adenoviral vectors: a new paradigm for cancer therapy
Investigator(s):	Jin D
Department:	Biochemistry
Source(s) of Funding:	Seed Funding Programme for Applied Research
Start Date:	06/2009
Completion Date:	05/2010

Abstract:

Please see uploaded file for details.

Project Title:	Knockdown of miR-BART5 in Epstein-Barr Virus-infected Cells by RNA Decoys Delivered through Novel Adenoviral Vectors: a New Paradigm for Cancer Therapy
Investigator(s):	Jin D, Kok KH, Lei T
Department:	Biochemistry
Source(s) of Funding:	Innovation and Technology Support Programme (Tier 3)
Start Date:	01/2010

Abstract:

Technological challenges: 1) Optimization of miR-BART5-inhibiting RNA decoys - Whereas the proofs of the principle that RNA decoys or sponges can be used to inhibit miRNA activity specifically have been provided by Nobel Prize laureate Phil Sharp and others since 2007, RNA decoys specifically targeting miR-BART5 of EBV must be further optimized if they are to be used in targeted therapy of NPC. 2) Delivery of RNA decoys into EBV-infected cancer cells - Phil Sharp and others have shown that RNA decoys chemically synthesized or delivered through lentiviral vectors can effectively inhibit their miRNA targets. However, how RNA decoys could be delivered specifically to cancer cells, but sparing the normal cells, remains a great technological challenge in the field. Specific targeting of EBV-infected cancer cells is therefore one of the most critical issues in targeted therapy of NPC. Objectives: 1) To develop cell-based assays to analyze the activity of anti-miR-BART5 RNA decoys 2) To construct Ad-EBER2 vectors and characterize them for driving the expression of RNA decoys in EBV+ cells 3) To optimize the RNA decoys expressed from Ad-EBER2 for most effective knockdown of miR-BART5 in EBV+ cells 4) To assess the additive or synergistic effects of RNA decoys and other agents on EBV+ cells 5) To determine the anti-tumour effects of RNA decoys in nude mice

Project Title:	Identification and characterization of the cellular targets of miR-BART5, an Epstein-Barr virus-encoded microRNA
Investigator(s):	Jin D, Kok KH, Tsao GSW
Department:	Biochemistry
Source(s) of Funding:	General Research Fund (GRF)
Start Date:	01/2010

Abstract:

1) Validation of MICB targeting by miR-BART5: a) Modulation of MICB expression by miR-BART5; b) Underexpression of MICB in EBV+ epithelial cells and tissues; c) Roles of miR-BART5 in immune evasion; 2) Is miR-BART5 a viral ortholog of cellular miR-18a? a) Targeting of common cellular transcripts by miR-BART5 and miR-18a; b) Sequence determinants of miR-BART5 and miR-18a targeting; c) Roles of miR-BART5 and miR-18a in cell survival, immune evasion and tumorigenesis; 3) Identification and characterization of additional cellular targets of miR-BART5: a) Bioinformatic analysis; b) Whole-genome approach of miR-BART5 target screening; c) Validation and further characterization of selected targets.

Project Title:	Knockdown of miR-BART5 in Epstein-Barr Virus-infected Cells by RNA Decoys Delivered through Novel Adenoviral Vectors: a New Paradigm for Cancer Therapy
Investigator(s):	Jin D
Department:	Biochemistry
Source(s) of Funding:	Innovation and Technology Fund Internship Programme
Start Date:	01/2010

Abstract:

Project Title:	Knockdown of miR-BART5 in Epstein-Barr Virus-infected Cells by RNA Decoys Delivered through Novel Adenoviral Vectors: a New Paradigm for Cancer Therapy
Investigator(s):	Jin D
Department:	Biochemistry
Source(s) of Funding:	Innovation and Technology Fund Internship Programme
Start Date:	01/2010

Abstract:

Project Title:	Regulatory roles of dsRNA-binding proteins PACT and TRBP in antiviral innate immunity
Investigator(s):	Jin D, Kok KH
Department:	Biochemistry
Source(s) of Funding:	Seed Funding Programme for Basic Research
Start Date:	03/2010

Abstract:

Antiviral innate immunity constitutes the first-line defense against viral infection and this is driven by the recognition of pathogen-associated molecular patterns on viruses by cellular pattern-recognition receptors. Type I interferons (IFNs) are important effectors in antiviral innate immunity, but the cellular signaling pathways leading from viral infection to activation of type I IFN production have just begun to be understood in recent years. The prototypic cytoplasmic sensor of viral nucleic acids known as retinoid acid-inducible gene I (RIG-I) was identified in 2004. Detection of viral double-stranded RNA (dsRNA) by RIG-I generates the activation signal adapted through a mitochondrial protein termed virus-induced signaling adaptor (VISA) to downstream kinases, which catalyze the phosphorylation of transcription factors called IFN regulatory factor (IRF) 3 and IRF7, leading ultimately to the activation of type I IFN promoters. We are interested in the identification and characterization of additional cellular and viral regulators of type I IFN production. Our recent findings suggest a new mechanism by which severe acute respiratory syndrome coronavirus circumvents the production of type I IFNs by impeding the formation of a functional IRF kinase complex through the viral matrix (M) protein (Siu et al., J. Biol. Chem., 2009). On the other hand, we have demonstrated that dsRNA binding protein PACT forms a complex with Dicer and TRBP to facilitate production of small interfering RNAs (Kok et al., J. Biol. Chem., 2007). Both Dicer and RIG-I have a DExD/H box RNA helicase domain. In addition, RIG-I homolog in C. elegans physically interacts with PACT homolog RDE4. This prompted us to investigate the interaction between PACT and RIG-I in human cells. Indeed, our preliminary findings indicate that PACT interacts with RIG-1 and potently stimulates RIG-I-induced activation of IFN-beta promoter. TRBP is a paralog of PACT. PACT and TRBP are highly homologous, but exert opposite effects on PKR. In this project we will characterize the roles of human PACT and TRBP in RIG-I-mediated activation of antiviral innate immunity. Specifically, we will verify the interaction between PACT/TRBP and RIG-I using co-fractionation and fluorescence resonance energy transfer technology. We will also define the interaction domains and assess the influence of dsRNA on PACT/TRBP-RIG-I interaction. Next we will investigate the mechanism of PACT/TRBP-mediated regulation of RIG-I by determining the influence of Dicer, dsRNA, and dsRNA-dependent protein kinase PKR on PACT/TRBP-RIG-I interaction as well as the influence of PACT/TRBP on RIG-I-dependent ATPase/helicase activity and complex formation. Our work will provide full characterization of PACT/TRBP-mediated regulation of RIG-I-mediated antiviral response. Our study addresses an important question in molecular immunology and virology. By deriving the new concept that the function of virus sensor RIG-I requires a dsRNA-binding protein partner, our work will substantially advance our understanding of virus-host interaction, particularly in the regard of cellular regulation of type I IFN production. Our findings will not only provide new avenues for studying viral and cellular regulators of innate immunity, but might also reveal novel strategies for development of antiviral and immunomodulatory agents. Key issues and questions being addressed: Host cells sense invading viruses and mobilize innate immune response to counteract their infection. How viral infection leads to the production of type I interferons (IFNs) was elusive until the identification of a prototypic cytoplasmic sensor of viral dsRNA called RIG-I in 2004. The current model for RIG-I-mediated activation of IFN production suggests that RIG-I sends an activation signal to a mitochondrial protein named VISA, which in turn activates TANK-binding kinase 1 (TBK1) and IkB kinase i (IKKi) to phosphorylate and activate IFN regulatory factor (IRF) 3 and IRF7 for subsequent binding to type I IFN promoters. However, the action of RIG-I plausibly requires additional partners and modifiers which remain to be identified. RIG-I is a DExD/H box RNA helicase bearing structural similarity with Dicer, an RNase III-type nuclease required for the generation and function of small interfering RNAs (siRNA) and microRNAs (miRNAs). Interestingly, Drosophila Dicer-2 is essential for antiviral response. We and others have shown that human Dicer requires dsRNA binding protein (dsRBP) partners TRBP and PACT for optimal activity in RNA interference (RNAi). Thus, it will be of great interest to investigate whether a dsRBP partner might also be necessary for full activation of RIG-I in innate antiviral immune response. In this regard, our careful re-analysis of published data indicates that a previously unrecognized RIG-I homolog in C. elegans physically interacts with PACT homolog RDE4. Verification of the interaction between PACT/TRBP and RIG-I in mammalian cells is therefore the next important issue to be addressed experimentally. TRBP and PACT are structurally related proteins capable of forming a complex, but they exert opposite regulatory activities on dsRNA-dependent protein kinase PKR, an IFN-induced enzyme that plays important roles in innate antiviral immunity, translational control and cell signaling. Although PACT is a potent activator of PKR, many of its activities are thought to be mediated through PKR-independent mechanisms that are poorly understood. Thus, identification and characterization of PKR-independent partners of PACT will shed mechanistic light on the function of this pleiotropic regulatory protein in antiviral innate immunity. Unifying hypothesis: PACT is a functional dsRBP partner of RIG-I in innate antiviral immunity. PACT interacts with and potently activates RIG-I leading ultimately to the induction of type I IFN expression. Plausibly through an interaction with PACT, TRBP inhibits RIG-I activity. The roles of PACT and TRBP might be relevant to viral infection, stress response and signal transduction. Objectives: 1) Verification of the interaction between PACT/TRBP and RIG-I: a) Biochemical co-fractionation analysis of protein complexes; b) Fluorescence resonance energy transfer analysis of intracellular interactions; c) Mapping of interaction domains in PACT/TRBP and RIG-I; d) Influence of dsRNA on PACT/TRBP-RIG-I interaction 2) Mechanism of PACT-mediated activation of RIG-I: a) Full characterization of PACT/TRBP-mediated regulation of RIG-I; b) Dependence of RIG-I activation on PKR and Dicer; c) Roles of dsRNA in PACT-induced activation of RIG-I; d) Influence of PACT on ATPase/helicase activity of RIG-I; e) Influence of PACT/TRBP on the formation of RIG-I complex

Project Title:	AICR 30th Anniversary Conference Targeting of INTS6 tumor suppressor by an Epstein-Barr virus-encoded microRNA
Investigator(s):	Jin D
Department:	Biochemistry
Source(s) of Funding:	URC/CRCG - Conference Grants for Teaching Staff
Start Date:	04/2010
Completion Date:	04/2010

Abstract:

N/A

List of Research Outputs

Chan C.P., Mak T.Y., Chin K.T., Ng I.O.L. and Jin D., N-linked glycosylation is required for optimal proteolytic activation of membrane-bound transcription factor CREB-H., J. Cell Sci.. The Company of Biologists, 2010, 123: 1438-1448.

Chan C.P., Mak T.Y., Chin K.T. and Jin D., Requirement of N-linked Glycosylation in the Luminal Domain for Optimal Proteolytic Activation of Liver-enriched transcription Factor CREB-H, 14th Research Postgraduate Symposium, December 2 & 3, 2009, The University of Hong Kong. 2009.

Chim J.C.S., Wong K.Y., Qi Y., Foong F., Lam W.L., Wong L.G., Jin D., Costello J.F. and Liang R.H.S., Epigenetic inactivation of the miR-34a in hematological malignancies, Carcinogenesis. 2010, 31(4): 745-50.

Chun C.S. and Jin D., Ubiquitin-dependent regulation of translesion polymerases., Biochem Soc Trans. 2010, 38: 110-115.

Jin D., Deputy Editor-in-Chief, The Bioscience Journal (The official jornal of the Society for Chinese Bioscientists in America). Biomed Central, 2010.

Jin D., Editorial Board Member, Oligonucleotides (The Official Journal of Oligonucleotide Therapeutics Society). Mary Ann Liebert, Inc., 2009.

Jin D., Editorial Board Member, World Journal of Hepatology. Beijing, Beijing Baishideng BioMed Scientifc Co., Ltd.,, 2009.

Jin D., Editorial Board member, World Journal of Biological Chemistry. Beijing, Baishideng Publishing, 2009.

Jin D., Inhibition of antiviral innate immunity by influenza virus NS1 protein., Area of Excellence Symposium – Should we target the virus, the cell or both? – Vaccine Development and Systems Biology. The university of Hong Kong, Hong Kong, June 3, 2010. .

Jin D., Modulation of innate antiviral immunity by influenza virus NS1 protein., The University of Hong Kong – Xiamen University – National Taiwan University Joint Scientific Symposium on Emerging infectious Diseases, Xiamen, September 18-19, 2009.. 2009.

Jin D., Viral and Cellular Regulators of Type I Interferon Production, Frontiers Biomedical Research, HKU. December 4. 2009.

Kok K.H., Lei T. and Jin D., siRNA and shRNA screens advance key understanding of host factors required for HIV-1 replication., Retrovirology. 2009, 6: 78.

Lam H.C., Jin D. and Song Y., Ptpn21, a Positive Mediator which Promotes ErbB4 and Suppresses Cell survival, 14th Research Postgraduate Symposium, December 2 & 3, 2009, The University of Hong Kong. 2009.

Mak T.Y., Chan C.P. and Jin D., Differential stability and transcriptional activity of CREB3 subfamily transcription factors, 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium, CUHK, Hong Kong, 15 May,. 2010.

Ng M.H., Ng R.K., Kong C.T., Jin D. and Chan L.C., Activation of Ras-dependent Elk-1 activity by MLL-AF4 family fusion oncoproteins, Experimental Hematology. 2010, 38: 481-488.

Ng M.H., Ng R.K., Kong C.T., Jin D. and Chan L.C., Activation of Ras-dependent Elk-1 activity by MLL-AF4 family fusion oncoproteins, Experimental Hematology. ELSEVIER, 2010, 38: 481-488.

Ng M.H., Siu K.L., Kok K.H., Ching Y.P. and Jin D., MIP-T3 is a novel inhibitor in TBK1/IRF3-dependent antiviral response. , 28th Annual meeting of the American Society for Virology. 2009.

Ng M.H., Siu K.L., Ching Y.P. and Jin D., MIP-T3 is a novel inhibitor in TBK1/IRF3-dependent antiviral response, The American Society for Virology 28th Annual Meeting, University of British Columbia, Vancouver, BC, Canada. 2009.

Siu K.L., Chan C.P., Chan C.S., Zheng B. and Jin D., Severe acute respiratory syndrome coronavirus nucleocapsid protein does not modulate transcription of human FGL2 gene., J. Gen. Virol.. 2009, 90: 2107-2113.

Tang V.H.M., Siu K.L., Wong C.M. and Jin D., Loss of Yeast Peroxiredoxin Tsa1p Induces Genome Instability through Activation of the DNA Damage Checkpoint and Elevation of dNTP Levels, In: Orna Cohen-Fix, PLoS Genetics. 2009, 5(10): e1000697.

Tang V.H.M., Kong K.Y., Wong G.W., Qiu H.F., Jin D. and Hinnebusch A.G., Yeast arginine methyltransferase Hmt1p is required for transcription elongation and termination, Mechanisms of Eukaryotic Transcription, Cold Spring Harbor Laboratory. 2009.

Wong C.M., Tang V.H.M., Kong K.Y., Wong G.W., Qiu H., Jin D. and Hinnebusch A.G., Yeast arginine methyltransferase Hmt1p regulates transcription elongation and termination by methylating Npl3p, Nucleic Acids Research. 2010, 38(7): 2217-2228.

Wong K.Y., Jin D., Liang R.H.S. and Chim J.C.S., Epigenetic silencing of miR-203 is a disease initiation event of multiple myeloma, European Association of Cancer Research, Norway (Poster). 2010.

Zhang H., Wang Y., Wu H., Jin D., Wen Y. and Zheng B., The Y271 And I274 Amino Acids In Reverse Transcriptase Of Human Immunodeficiency Virus-1 Are Critical To Protein Stability, In: Olivier Schwartz, Plos One. 2009, 4.

Researcher : Jin G

List of Research Outputs

Jin G., MT1-MMP Regulates Early Lymphocyte Development through Notch Signaling, PhD Thesis, 2010.

Zhou Z., Jin G. and Chan J.K.M., Mt1-mmp Regulates Fgf Signaling In Osteogenesis, Gordon Research Conference-Matelloproteinses. Les Diablerets, Switzerland, 2009.

Researcher : Jin Y

Project Title:	Role of a small noncoding RNA SroH in acid resistance in Escherichia coli
Investigator(s):	Jin Y, Huang J
Department:	Biochemistry
Source(s) of Funding:	Small Project Funding
Start Date:	08/2008
Completion Date:	08/2009

Abstract:

Commensal and pathogenic Escherichia coli must survive the acidic conditions of the stomach, where the pH is normally between 2 and 3, before they can colonize a mammalian host. The bacteria possess three strategies to overcome the acidic stress, including glutamate and arginine decarboxylase systems and a glucose-catabolite-repressed system (Castanie-Cornet et al., 1999; Lin et al., 1996; Richard et al., 2003). Small noncoding RNAs (sncRNA) are a set of short RNAs (between 50 and 250nt long) that are not translated into proteins. sncRNAs basepairing with mRNA targets affect either the stability or translation of the mRNAs, and thereby act as regulatory factors in a variety of stress responses (Storz et al., 2004). However, the roles of sncRNAs in the network of acid resistance remain elusive. We have established a sncRNA knockout library of E. coli K12 MG1655. Totally eighty-one knockout mutants lacking sncRNAs identified prior to 2006 were included in the library. By screening for their ability to survive at acidic conditions (pH<2.5), we have found that E. coli cells lacking sncRNA SroH survive better than wild type at low pH, indicating that SroH is a negative regulator of acid resistance in E. coli. Thus, the objectives of the present study are: a. To confirm the role of SroH in acid resistance in E. coli by gain-of-function assays b. To identify the mRNA targets of SroH for regulation of acid resistance c. To elucidate the possible roles of SroH in H-NS- and RpoS-dependent regulatory cascade responsible for acid resistance

List of Research Outputs

Jin Y., Watt R.M., Danchin A. and Huang J., Use of a riboswitch-controlled conditional hypomorphic mutation to uncover a role for the essential csrA gene in bacterial autoaggregation, Journal of Biological Chemistry. 2009, 284: 28738-28745.

Mochon A.B., Jin Y., Kayala M.A., Wingard J.R., Clancy C.J., Nguyen M.H., Felgner P., Baldi P. and Liu H., Serological profiling of a Candida albicans protein microarray reveals permanent host-pathogen interplay and stage-specific responses during candidemia, PLos Pathogens. 2010, 6: e1000827.

Researcher : Kao PYP

List of Research Outputs

Kao P.Y.P., Genetic Association Studies of Lumbar Disc Degeneration (LDD), PhD Thesis, 2010.

Kao P.Y.P., Yang D., Lau L.S., Tsui H.W., Hu L., Dai J., Chan M.P., Chan C.M., Wang P., Zheng B., Sun J., Huang J., Madar J., Chen G., Chen H., Guan Y. and Yuen K.Y., Identification of Influenza a Nucleoprotein as an Antiviral Target, Nature Biotechnology. 2010, 28: 600-608.

Researcher : Kao YPP

List of Research Outputs

Kao Y.P.P., Chan D., Cheah K.S.E., Cheung K.M.C., Ho D.W.H., Karppinen J., Leong J.C.Y., Yip S.P., Song Y. and Sham P.C., Genome-wide Association Study of Degenerative Disc Disease (DDD), 14th Research Postgraduate Symposium, December 2 & 3, 2009, The University of Hong Kong. 2009.

Researcher : Kimura M

List of Research Outputs

Tanner J.A., Zheng B., Lin Y., Kimura M., Lui E.L.H. and Shum K.T., Selection, validation and delivery of DNA aptamers against infectious disease targets. , 5th Annual Meeting of the Oligonucleotide Therapeutics Society, Fukuoka, Japan . 2009.

Researcher : Kok KH

Project Title:	Characterization of the interaction between RNA binding proteins TRBP and hnRNP A1 in mammalian cells
Investigator(s):	Kok KH, Jin D
Department:	Biochemistry
Source(s) of Funding:	Small Project Funding
Start Date:	09/2008
Completion Date:	02/2010

Abstract:

Project Objectives: 1) Characterization of TRBP-hnRNP A1 interaction in cultured mammalian cells 2) Determination of the functional implications of TRBP-hnRNP A1 interaction in miRNA-mediated translational repression Major hypothesis: Heterogeneous nuclear ribonucleoprotein (hnRNP) A1 is a multifunctional RNA binding protein critically involved in mRNA splicing, pre-mRNA export, and translational control. hnRNP A1 can shuttle from the nucleus to the cytoplasm and it binds to a subset of mRNAs to regulate their translation. In particular, the binding of hnRNP A1 to its target sequence (TAGGGA/T) located in the 3'UTR of the mRNA increases protein expression, while knockdown of hnRNP A1 or forced expression of dominant negative hnRNP A1 mutant inhibits mRNA translation. However, the exact mechanism by which hnRNP A1 protects the bound mRNA is not known. TRBP is a dsRNA-binding protein and its association with Dicer is required for miRNA biogenesis and function. We have recently demonstrated a new model for TRBP function in which TRBP forms a triple complex with PACT and Dicer to mediate RNA interference (Kok et al., 2007). In addition, in our pilot study based on proteomic analysis of TRBP-binding proteins, we have identified hnRNP A1 as a novel binding partner of TRBP. In mammalian cells, primary miRNA (pri-miRNA) is processed by Drosha and its dsRNA-binding protein partner DRGC8 before export to the cytoplasm. The processed pre- miRNA is further cleaved by Dicer/TRBP/PACT in the cytoplasm to generate mature miRNA which incorporates into RISC complex containing TRBP, Dicer and Ago2, to mediate translational repression of the target mRNA. The interaction between hnRNP A1 and TRBP raises the question as to whether hnRNP A1 might play a role in TRBP-regulated miRNA biogenesis and function. In this study, we will systematically address the interaction between hnRNP A1 and TRBP. In addition, we will also shed light on the functional implication of this pair of protein-protein interaction in miRNA biogenesis and function. Key issues and problems being addressed: 1) Characterization of hnRNP A1-TRBP interaction: Verification and characterization of hnRNP A1-TRBP interaction with different methods provide the foundation for further functional study. 2) Functional implication of the hnRNP A1-TRBP interaction: We will focus primarily on the influence of hnRNP A1 binding to 3 UTR of mRNA on miRNA-mediated translational repression, a process that requires TRBP

List of Research Outputs

Kok K.H., Lei T. and Jin D., siRNA and shRNA screens advance key understanding of host factors required for HIV-1 replication., Retrovirology. 2009, 6: 78.

Ng M.H., Siu K.L., Kok K.H., Ching Y.P. and Jin D., MIP-T3 is a novel inhibitor in TBK1/IRF3-dependent antiviral response. , 28th Annual meeting of the American Society for Virology. 2009.

Researcher : Kong CT

List of Research Outputs

Ng M.H., Ng R.K., Kong C.T., Jin D. and Chan L.C., Activation of Ras-dependent Elk-1 activity by MLL-AF4 family fusion oncoproteins, Experimental Hematology. 2010, 38: 481-488.

Ng M.H., Ng R.K., Kong C.T., Jin D. and Chan L.C., Activation of Ras-dependent Elk-1 activity by MLL-AF4 family fusion oncoproteins, Experimental Hematology. ELSEVIER, 2010, 38: 481-488.

Researcher : Kong KY

List of Research Outputs

Kong K.Y., University Postgraduate Fellowship, The University of Hong Kong. 2009.

Tang V.H.M., Kong K.Y., Wong G.W., Qiu H.F., Jin D. and Hinnebusch A.G., Yeast arginine methyltransferase Hmt1p is required for transcription elongation and termination, Mechanisms of Eukaryotic Transcription, Cold Spring Harbor Laboratory. 2009.

Wong C.M., Tang V.H.M., Kong K.Y., Wong G.W., Qiu H., Jin D. and Hinnebusch A.G., Yeast arginine methyltransferase Hmt1p regulates transcription elongation and termination by methylating Npl3p, Nucleic Acids Research. 2010, 38(7): 2217-2228.

Researcher : Kwok STC

List of Research Outputs

Kwok S.T.C., Wu M.H., Herve A., Nieto M.A. and Cheung M.C.H., The role of SoxE transcription factors in melanoma development, 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium, CUHK, Hong Kong, 15 May. 2010.

Researcher : Kwong WH

List of Research Outputs

Kwong W.H., Functional analyses on TGFβ/BMP Signaling and Type II/A Procollagen in Inner Ear Development, PhD Thesis. 2009.

Researcher : Lam HC

List of Research Outputs

Lam H.C., Functional characterization of tyrosine phosphatase non-receptor 21, a novel modulator of ErbB4/NRG3, M.Phil Thesis , 2010.

Lam H.C., Jin D. and Song Y., Ptpn21, a Positive Mediator which Promotes ErbB4 and Suppresses Cell survival, 14th Research Postgraduate Symposium, December 2 & 3, 2009, The University of Hong Kong. 2009.

Researcher : Lam HC

List of Research Outputs

Lam H.C., Functional characterization of tyrosine phosphatase non-receptor 21, a novel modulator of ErbB4/NRG3, M.Phil Thesis , 2010.

Researcher : Lam KY

List of Research Outputs

Lam K.Y., Differential Regulation of FOXM1 isoforms by Raf/MEK/ERK Signaling, M.Phil Thesis, 2010.

Lam K.Y. and Yao K.M., Differential Regulation of FoxM1 Isoforms by Raf/MEK/ERK Signaling, 14th Research Postgraduate Symposium, December 2 & 3, 2009, The University of Hong Kong. 2009.

Researcher : Lam KY

List of Research Outputs

Lam K.Y., Differential Regulation of FOXM1 isoforms by Raf/MEK/ERK Signaling, M.Phil Thesis, 2010.

Lam K.Y. and Yao K.M., Differential Regulation of FoxM1 Isoforms by Raf/MEK/ERK Signaling, 14th Research Postgraduate Symposium, December 2 & 3, 2009, The University of Hong Kong. 2009.

Researcher : Lam VMS

Project Title:	18th International Congress of Biochemistry and Molecular Biology Beyond the Human Glucose-6-phosphate Dehydrogenase (G6PD) Gene: Protein Structure and Function Analyses
Investigator(s):	Lam VMS
Department:	Biochemistry
Source(s) of Funding:	URC/CRCG - Conference Grants for Teaching Staff
Start Date:	07/2000

Abstract:

N/A

Project Title:	HUGO's Seventh International Human Genome Meeting, HGM2002 Post-Genomic Glucose-6-phosphate Dehydrogenase (G6PD): Different Mechanisms for Enzyme Deficiency from Structural and Kinetic Analyses
Investigator(s):	Lam VMS
Department:	Biochemistry
Source(s) of Funding:	URC/CRCG - Conference Grants for Teaching Staff
Start Date:	04/2002

Abstract:

N/A

Project Title:	7th International Symposium on Mutations in the Human Genome Multiplex Detection: a Rapid and Reliable Method to Screen Common Variations in the Glucose-6-phosphate Dehydrogenase Gene
Investigator(s):	Lam VMS
Department:	Biochemistry
Source(s) of Funding:	URC/CRCG - Conference Grants for Teaching Staff
Start Date:	07/2003

Abstract:

N/A

Researcher : Lee KS

List of Research Outputs

Lee K.S., Lam T.K., Song Y., Cheah K.S.E., Cheung K.M.C. and Chan D., In vivo study of Asporin, a genetic risk factor for Osteoarthritis and degenerative disc disease, 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium, CUHK, Hong Kong, 15 May,. 2010.

Researcher : Lei IL

List of Research Outputs

Lei I.L., Gao X.L., Wang Z. and Sham M.H., BAF250a-containing complex is required for secondary heart field development, 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium, CUHK, Hong Kong, 15 May,. 2010.

Researcher : Lei T

Project Title:	A→I editing of Epstein-Barr virus-encoded BART microRNAs and its impact on microRNA biogenesis
Investigator(s):	Lei T, Jin D
Department:	Biochemistry
Source(s) of Funding:	Small Project Funding
Start Date:	11/2009

Abstract:

Epstein-Barr virus (EBV) in the family of Herpesviridae is one of the most successful human viruses, infecting greater than 90% of the world’s adult population [1]. EBV is also the first human virus found to express microRNAs (miRNAs), which are gene regulatory RNAs of ~22-nucleotides in length and regulate gene expression by repressing translation or reducing mRNA stability, and these microRNAs emerged as important regulators of viral life cycle, virus-host cell interactions and EBV-associated carcinogenesis [2]. Although insights into the regulatory function of miRNAs are beginning to emerge, much less is known about the regulation of miRNA expression and activity.BART miRNAs are initially transcribed by RNA polymerase II as long primary transcript (BART RNA or pri-miRNA) and processed first in the nucleus by the RNase enzyme complex, Drosha-DGCR8, into one or more miRNA precursors (pre-miRNA). Pre-miRNA are about 65-85 bp long and folded into stem-loop structure that is exported to the cytoplasm by the nuclear export factor exportin-5 coupled functionally with RanGTP. Cytoplasmic Dicer, together with TRBP, processes the pre-miRNAs into ~22 bp mature miRNAs. After integration into the RNA-induced silencing complex (RISC), miRNAs block translation of specific mRNAs containing partially complementary target sequence located in the 3’UTR5. These maturation steps are believed to be affected by different mechanisms. Post-transcriptional regulation of miRNA processing can be observed in cancer cells and might explain the aberrant miRNA expression pattern [3,4]. RNA editing is a post-transcriptional process that changes the nucleotide sequence of an RNA transcript [5]. Emerging evidence suggests that A→I RNA editing can modulate RNAi. Many proteins that are involved in miRNA processing, such as Dicer, Drosha, DGCR8 and TRBP, contain double strand RNA binding domain (dsRBD), as do ADARs (adenosine deaminases acting on RNA), the enzymes for A→I editing. Because dsRBPs are not sequence-specific, any dsRNAs that are recognized by the RNAi mechanism are also potential targets for A→I RNA editing by ADARs. Recent studies showed that precursor RNAs of certain miRNAs indeed undergo A→I RNA editing [6,7]. A systematic survey of human pri-miRNA sequences identified A-I RNA editing sites in ~6% of all pri-miRNAs examined [8]. However, in vitro editing studies of randomly selected pri-miRNAs predict that as many as 50% of all pri-miRNAs might have specific A→I RNA editing sites [6]. RNA editing of primary transcripts by ADAR modifies adenosine (A) into inosine (I). Because the base-pairing properties of inosine are similar to those of guanosine (G), A-to-I editing of miRNA precursors may change their sequence, base-pairing and structural properties and can influence their further processing as well as their target recognition abilities. Of note, EBV encoded miRNAs appear to be processed by the same Drosha and Dicer dependent pathway used by the majority of cellular miRNAs. Does RNA editing also affect EBV miRNA processing? The frequency of EBV miRNA editing and the fate of edited pri-miRNAs remain largely unknown. In this study, we set out to investigate the A→I RNA editing of EBV miRNAs and its impact on miRNA biogenesis. Project objectives A) Identification of A→I edited sites in EBV miRNAs B) Verification of A→I editing of EBV miRNAs C) Influence of A→I editing on biogenesis of EBV miRNAs 1. Crawford, D.H. Biology and disease associations of Epstein-Barr virus. Philos Trans R Soc Lond B Biol Sci 356, 461-73 (2001). 2. Gottwein, E. & Cullen, B.R. Viral and cellular microRNAs as determinants of viral pathogenesis and immunity. Cell Host Microbe 3, 375-87 (2008) 3. Lee, E.J. et al. Systematic evaluation of microRNA processing patterns in tissues, cell lines, and tumors. RNA 14, 35-42 (2008). 4. Valencia-Sanchez, M.A., Liu, J., Hannon, G.J. & Parker, R. Control of translation and mRNA degradation by miRNAs and siRNAs. Genes Dev 20, 515-24 (2006). 5.Gott, J.M. & Emeson, R.B. Functions and mechanisms of RNA editing. Annu Rev Genet 34, 499-531 (2000). 6. Luciano, D.J., Mirsky, H., Vendetti, N.J. & Maas, S. RNA editing of a miRNA precursor. RNA 10, 1174-7 (2004). 7. Yang, W. et al. Modulation of microRNA processing and expression through RNA editing by ADAR deaminases. Nat Struct Mol Biol 13, 13-21 (2006). 8. Blow, M.J. et al. RNA editing of human microRNAs. Genome Biol 7, R27 (2006).

List of Research Outputs

Kok K.H., Lei T. and Jin D., siRNA and shRNA screens advance key understanding of host factors required for HIV-1 replication., Retrovirology. 2009, 6: 78.

Researcher : Leong JCY

List of Research Outputs

Kao Y.P.P., Chan D., Cheah K.S.E., Cheung K.M.C., Ho D.W.H., Karppinen J., Leong J.C.Y., Yip S.P., Song Y. and Sham P.C., Genome-wide Association Study of Degenerative Disc Disease (DDD), 14th Research Postgraduate Symposium, December 2 & 3, 2009, The University of Hong Kong. 2009.

Researcher : Leung C

List of Research Outputs

Zhang M., Leung C., Lui V.C.H., Tam P.K.H. and Sham M.H., Sox10 affects enteric neural crest cells migration in a Sox10NGFP/+ mutant , 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium, CUHK, Hong Kong, 15 May,. 2010.

Zhang M., Leung C., Lui V.C.H., Tam P.K.H. and Sham M.H., Sox10 is required for proliferation and migration of enteric neural crest stem cells, International Society for Stem Cell Research, 8th Annual Meeting, Moscone West, San Francisco, CA, USA, June 16-19, 2010.

Zhang M., Leung C., Lui V.C.H., Tam P.K.H. and Sham M.H., Sox10 mediates proliferation and migration behavior of enteric neural crest cells, 43rd Annual Meeting for the Japanese Society of Developmental Biologists, Jointly Sponsored by the Asia-Pacific Developmental Biology Network, Kyoto, Japan, 20-23 June. 2010.

Researcher : Leung CM

List of Research Outputs

Shum K.T., Chan C., Leung C.M. and Tanner J.A., Aptamer-based Inhibitor of Sclerostin for Osteoporosis Therapy, 14th Research Postgraduate Symposium, December 2 & 3, 2009, The University of Hong Kong. 2009.

Shum K.T., Chan C.S.L., Leung C.M. and Tanner J.A., Aptamer-mediated inhibition of sclerostin for osteoporosis therapy. , 5th Annual Meeting of the Oligonucleotide Therapeutics Society, Fukuoka, Japan . 2009.

Shum K.T., Chan C.S.L., Leung C.M. and Tanner J.A., G-Quadruplex Aptamers Inhibit Sclerostin for Osteoporosis Therapy, CUHK Croucher Advanced Study Institute “Structure-Based Screening and Design of Ligands for Protein Targets” . 2009.

Researcher : Leung CM

List of Research Outputs

Researcher : Leung KKH

List of Research Outputs

Ho B.S., Cheng C.C.W., Leung K.K.H. and Cheah K.S.E., The role of Sox2 and Sox8 in mouse hair development, 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium, CUHK, Hong Kong, 15 May,. 2010.

Leung Y.L., Gao B., Melhado I.G., Leung K.K.H., Wynn S.L., Chan D. and Cheah K.S.E., Sox9 mediates gene expression specificity in growth plate chondrocytes via concomitant positive and negative regulation, 55th Annual Meeting of the Orthopaedic Research Society, New Orleans, USA. 2010.

Researcher : Leung OYV

List of Research Outputs

Leung O.Y.V., Targeting Unopposed Neutrophil Elastase in Chronic Respiratory Inflammation with Heparin Oligosaccharides, MPhil Thesis. 2009.

Shum D.K.Y., Ip M.S.M., Chan C.H. and Leung O.Y.V., Compositions and Methods for Treating chronic Respiratory Inflammation. U.S. Provisional Application No. 61/308,597 filed on Feb 26, 2010 with the U.WS. Patent and Trademark Office. , 2010.

Researcher : Leung VOY

List of Research Outputs

Chan C.H., Leung V.O.Y., Lam C.L.D., Mak J.C.W., Freeman C., Ip M.S.M. and Shum D.K.Y., Sulfated maltoheptaose reduces neutrophilic airway inflammation in a smoking rat model of chronic obstructive pulmonary disease , Fifth International Symposium on Healthy Aging: “Is Aging a Disease?” 6-7 March 2010 . 2010.

Researcher : Leung WL

List of Research Outputs

Gao Y., Leung W.L., Wong S.Y.Y., Tam P.P.L., Chan D. and Cheah K.S.E., Type IIA Procollagen: A Positive Regulator of Nodal Signaling During Early Vertebrate Embryogenesis, 14th Research Postgraduate Symposium, December 2 & 3, 2009, The University of Hong Kong. 2009.

Gao Y., Leung W.L., Wong Y.Y., Chan D., Tam P.P.L. and Cheah K.S.E., Type IIA procollagen: A positive regulator of nodal signaling during embryogenesis, 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium, CUHK, Hong Kong, 15 May. 2010.

Researcher : Leung WL

List of Research Outputs

Researcher : Li J

List of Research Outputs

Li J., Bone Fracture Healing in Laminopathy-based Premature Aging, PhD Thesis, 2010.

Li J., Lee Y.F. and Cheah K.S.E., Identification of regulatory elements directing control of Sox2 expression in mouse inner ear development, 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium, CUHK, Hong Kong, 15 May,. 2010.

Researcher : Li J

List of Research Outputs

Li J., Bone Fracture Healing in Laminopathy-based Premature Aging, PhD Thesis, 2010.

Researcher : Li L

List of Research Outputs

Lui E.L.H., Li L. and Tanner J.A., Impact of Extracellular Inorganic Polyphosphate on Osteoblast Proliferation, Differentiation and Apoptosis. , 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium . 2010.

Researcher : Li M

List of Research Outputs

Li M., Expression of Chondroitin Sulfotransferases in Relation to Cranial Motor Neuron Movements in the Embryonic hindbrain, PhD Thesis , 2010.

Researcher : Lie YS

List of Research Outputs

Lie Y.S., Chan W.C.W., Tsang K.Y., Cheah K.S.E. and Chan D., Impact of intracellular clearance of unfolded proteins on chondrocytes cell fate, 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium, CUHK, Hong Kong, 15 May,. 2010.

Researcher : Lin R

List of Research Outputs

Cui J., Wang Z., Cheng Q.N., Lin R., Zhang X., Leung P.S., Copeland N.G., Jenkins N.A., Yao K.M. and Huang J., Targeted Inactivation of KIF5B in Pancreatic Leads to Insulin Secretory Deficiency and Increased b-Cells Mass in vivo, 14th Research Postgraduate Symposium, December 2 & 3, 2009, The University of Hong Kong. 2009.

Lin R., Lau C.F., Wang J., Ni Y., Fung M.L. and Huang J., Reduced neuronal nitric oxide production in KIF5b-deficiency mice, 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium, CUHK, Hong Kong, 15 May,. 2010.

Researcher : Lin R

List of Research Outputs

Researcher : Liu B

Project Title:	Adult stem cell decline accelerates premature ageing in Zmpste24 deficient mice
Investigator(s):	Liu B, Zhou Z
Department:	Biochemistry
Source(s) of Funding:	Small Project Funding
Start Date:	08/2008

Abstract:

Hutchinson-Gilford progeria syndrome (HGPS) is a rare genetic disorder of early onset premature ageing, predominantly caused by de novo G608G mutation in LMNA gene. Mice harboring LmnaL530P/L530P mutation or deficient for Zmpste24, a metalloproteinase responsible for prelamin A maturation, recapitulate many of the premature ageing features found in HGPS. HGPS is characterized by a rapid functional deterioration in various tissues, which could probably be caused by accelerated senescence or/and adult stem cell depletion. Adult stem cells persist in the majority of adult tissues and are capable of self-replenishment and differentiation into different specialized cell types. During ageing process, somatic cells are constantly exposed to a variety of exogenous and endogenous conditions that can lead to damages. To maintain tissue integrity, damaged cells can either be restored by damage repair machinery or, if the damage is too extensive, the cells may choose to become apoptotic or senescent, and eventually be eliminated or replaced by new ones differentiated from adult stem cells. Either accelerated senescence or exhaustion of adult stem cells could disrupt the homeostasis and lead to tissue degeneration. Adult stem cell decline either functionally or in number is also observed in and thought to be responsible for many degenerative diseases. We previously showed that HGPS skin fibroblasts and mouse embryonic fibroblasts (MEFs) derived from Zmpste24-/- embryos undergo earlier senescence due to genomic instability and hyper-activation of p53 pathways. Reducing prelamin A by Lmna heterozygosity ameliorates premature ageing and significantly extends the life span of Zmpste24-/- mice. Recently we and others demonstrated that adult stem cell decline or functional impairment was associated with premature ageing in Zmpste24-/- mice. However it is still unclear whether adult stem cell decline is a cause or a consequence of ageing. Interestingly, our preliminary results indicated that Lmna heterozygosity couldn’t ameliorate accelerated senescence in mouse embryonic fibroblasts (MEFs) though tissue senescence can be rescued in 6-month-old Lmna+/-Zmpste24-/- mice. We hypothesize that (1) reducing unprocessed prelamin A could restore or ameliorate adult stem cell decline, which could in turn replenish senescent cells inside the tissues; (2) Both adult stem cell decline and somatic cell senescence accelerate ageing in Zmpste24-/- mice. So the aim of this project is (1) to check whether half of prelamin A is still enough to accelerate senescence in somatic cells (i.e. MEFs); (2) to examine if Lmna heterozygosity can rescue adult stem cell decline to extend life span of Zmpste24-/- mice.

Project Title:	Stem Cells 2009 Oxidative stress-mediated accelerated adult stem cell decline contributes to premature ageing in Zmpste24–/– mice
Investigator(s):	Liu B
Department:	Biochemistry
Source(s) of Funding:	URC/CRCG - Conference Grants for Teaching Staff
Start Date:	11/2009

Abstract:

N/A

Researcher : Liu C

List of Research Outputs

Gao Y.P., Liu C., Chan Y.S. and He J.F., Contribution of thalamic reticular nucleus to the synchronization of spindle oscillation in the thalamocortical network, Neuroscience Bulletin (Suppl 1). [8th Biennial Conference of Chinese Society for Neuroscience. Guangzhou. Nov. 7-10, 2009]. 2009, 25: 353-354.

Liu C., Wang Z., Huen M.S.Y., Lu L., Liu D. and Huang J., Cell death caused by single-stranded oligodeoxynucleotides mediated targeted genomic sequence modification, Oligonucleotides. 2009, 19(3): 281-286.

Liu C., Fu X.F., Ren X.J., Chen G.H., Tang L.H., Hwa T.T. and Huang J., Formation of Novel Pattern by Programming Cell Motility, 14th Research Postgraduate Symposium, December 2 & 3, 2009, The University of Hong Kong. 2009.

Liu C., Fu X., Chen G., Tang L.H., Hwa T. and Huang J., Formation of novel pattern by programming cell motility, 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium, CUHK, Hong Kong, 15 May,. 2010.

Liu C., Gao Y.P., Gao L.X., Meng X.K., Yu Y.Q., Chan Y.S. and He J.F., The spindle oscillations in the thalamic reticular nucleus, auditory thalamus, and cortex, Neuroscience Bulletin (Suppl 1). [8th Biennial Conference of Chinese Society for Neuroscience. Guangzhou. Nov. 7-10, 2009]. 2009, 25: 353.

Xie L...X..., Durairajan S...S...K..., Lu J...H..., Liu C., Liu C., Kum W...F..., Wang Y., Koo I., Wu W...K..., Han D., Lao F., Huang J. and Li M., The effect of salvianolic acid B combined with laminar shear stress on TNF--stimulated adhesion molecule expression in human aortic endothelial cells, Clinical Hemorheology and Microcirculation. 2010, 44: 245–258.

Researcher : Liu D

List of Research Outputs

Liu C., Wang Z., Huen M.S.Y., Lu L., Liu D. and Huang J., Cell death caused by single-stranded oligodeoxynucleotides mediated targeted genomic sequence modification, Oligonucleotides. 2009, 19(3): 281-286.

Researcher : Lovell-Badge RH

List of Research Outputs

Cheah K.S.E., Au Y.K., Szeto Y.Y., Wynn S., Chan Y.S., Cheung K.M.C., Chan W.Y., Lovell-Badge R.H., Chan D. and Fritzsch B., Molecular and developmental insights into the pathogenesis of the SOX9Y440Xcampomelic dysplasia mutation, 16th International Society of Developmental Biologists Congress 6-10th September 2009 Edinburgh International Conference Centre, UK . 2009.

Researcher : Lu L

List of Research Outputs

Liu C., Wang Z., Huen M.S.Y., Lu L., Liu D. and Huang J., Cell death caused by single-stranded oligodeoxynucleotides mediated targeted genomic sequence modification, Oligonucleotides. 2009, 19(3): 281-286.

Lu L., Chan D., Cheah K.S.E. and Cheung M.C.H., Epigenetic Reprogramming of Fibroblasts into Osteo-chondroprogenitors, 14th Research Postgraduate Symposium, December 2 & 3, 2009, The University of Hong Kong. 2009.

Lu L., Cheah K.S.E., Chan D. and Cheung M.C.H., Epigenetic reprogramming of fibroblasts into osteo-chondroprogenitors, 43rd Annual Meeting for the Japanese Society of Developmental Biologists Kyoto, Japan, 20 June 2010 . 2010.

Lu S., Lu L., Guan X.Y. and Huang J., The Phenotype Study of Pdss2 Conditional Knockout Mouse, 14th Research Postgraduate Symposium, December 2 & 3, 2009, The University of Hong Kong. 2009.

Wang Y., Lu L., Wu M.H., Sham M.H., Chan D., Cheah K.S.E. and Cheung M.C.H., In vitro generation of Osteo-chondroprogenitors, 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium, CUHK, Hong Kong, 15 May,. 2010.

Researcher : Lu L

List of Research Outputs

Lu S., Lu L., Guan X.Y. and Huang J., The Phenotype Study of Pdss2 Conditional Knockout Mouse, 14th Research Postgraduate Symposium, December 2 & 3, 2009, The University of Hong Kong. 2009.

Researcher : Lu L

List of Research Outputs

Lu S., Lu L., Guan X.Y. and Huang J., The Phenotype Study of Pdss2 Conditional Knockout Mouse, 14th Research Postgraduate Symposium, December 2 & 3, 2009, The University of Hong Kong. 2009.

Researcher : Lu L

List of Research Outputs

Lu S., Lu L., Guan X.Y. and Huang J., The Phenotype Study of Pdss2 Conditional Knockout Mouse, 14th Research Postgraduate Symposium, December 2 & 3, 2009, The University of Hong Kong. 2009.

Researcher : Lu S

List of Research Outputs

Lu S., Phenotype analysis of Pdss2 conditional knockout mouse, PhD Thesis, 2010.

Lu S., Lu L., Guan X.Y. and Huang J., The Phenotype Study of Pdss2 Conditional Knockout Mouse, 14th Research Postgraduate Symposium, December 2 & 3, 2009, The University of Hong Kong. 2009.

Researcher : Lu S

List of Research Outputs

Lu S., Phenotype analysis of Pdss2 conditional knockout mouse, PhD Thesis, 2010.

Lu S., Lu L., Guan X.Y. and Huang J., The Phenotype Study of Pdss2 Conditional Knockout Mouse, 14th Research Postgraduate Symposium, December 2 & 3, 2009, The University of Hong Kong. 2009.

Researcher : Lui ELH

List of Research Outputs

Lui E.L.H., Shum K.T., Sam L., Wang Y., Watt R.M. and Tanner J.A., Aptamers Against Polyphosphate Kinase 2 (PPK2) From Mycobacterium Tuberculosis As A Potential Approach Of Novel Anti-Tuberculosis Therapy , CUHK Croucher Advanced Study Institute “Structure-Based Screening and Design of Ligands for Protein Targets” . 2009.

Lui E.L.H., Li L. and Tanner J.A., Impact of Extracellular Inorganic Polyphosphate on Osteoblast Proliferation, Differentiation and Apoptosis. , 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium . 2010.

Shum K.T., Lui E.L.H., Wang Y., Watt R.M. and Tanner J.A., Aptamer-mediated inhibition of Mycobacterium tuberculosis polyphosphate kinase 2. , 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium . 2010.

Tanner J.A., Zheng B., Lin Y., Kimura M., Lui E.L.H. and Shum K.T., Selection, validation and delivery of DNA aptamers against infectious disease targets. , 5th Annual Meeting of the Oligonucleotide Therapeutics Society, Fukuoka, Japan . 2009.

Researcher : Lui LHE

List of Research Outputs

Lui L.H.E., Shum K.T. and Tanner J.A., Effect of Inorganic Polyphosphate on Higher Eukaryotic Cells, 14th Research Postgraduate Symposium, December 2 & 3, 2009, The University of Hong Kong. 2009.

Researcher : Lui VCH

Project Title:	15th International Society of Development Biologists Congress (ISDB 2005) Perturbation of Hoxb5 Signalling in Vagal Neural Crest Cells Causes Defective Development of the Enteric Nervous System
Investigator(s):	Lui VCH
Department:	Surgery
Source(s) of Funding:	URC/CRCG - Conference Grants for Teaching Staff
Start Date:	09/2005

Abstract:

N/A

Project Title:	Investigation of cell autonomous function of Hedgehog signaling on vagal neural crest cells by conditional knockout of Smoothened in mice
Investigator(s):	Lui VCH, Ngan ESW, Tam PKH, Sham MH
Department:	Surgery
Source(s) of Funding:	General Research Fund (GRF)
Start Date:	01/2007
Completion Date:	12/2009

Abstract:

(1) Cross floxed Smo mice with a vagal NCC specific cre mouse strain (b3-IIIa-cre); (2) analyze the abnormal phenotypes in the developing ENs of the transgenic mutant mice; (3) analyze the effects of mesenchyme-derived factors on NCCs of the transgenic mutant mice.

Project Title:	Evaluation of HOXB5 as a Hirschsprung’s disease locus
Investigator(s):	Lui VCH, Garcia-Barcelo MM, Tam PKH
Department:	Surgery
Source(s) of Funding:	Seed Funding Programme for Basic Research
Start Date:	03/2008
Completion Date:	08/2009

Abstract:

Purposes: (1) To elucidate the mechanisms by which HOXB5 gene transcription is regulated (2) To evaluate HOXB5 as a novel Hirschsprung’s disease locus Key Issues: The enteric nervous system (ENS) comprises a network of neuronal ganglia and glia within the gut wall, which controls intestinal peristalsis. In mammals, vagal neural crest cells (NCC) migrate from the neural tube, enter the foregut and colonize the intestine, where they differentiate into neurons and glia (1). Defective ENS development results in fewer ganglia (hypoganglionosis), and/or absence of ganglia (aganglionosis) at the caudal-most gut in newborns with Hirschsprung’s disease (HSCR). Affected neonates develop a life-threatening condition of intestinal obstruction due to defective peristalsis and require surgery. HSCR causes considerable mortality and morbidity. There is a significant variation in the disease incidence among races with the highest incidence in Asians (2.8 per 10,000 life births). HSCR has a complex pattern of inheritance and manifests with incomplete penetrance and variable length of the aganglionic segment (2), indicating that HSCR results from the interaction of several genes. ENS development is regulated by molecular signals from within the NCC and the intestinal environment. The receptor tyrosine kinase gene RET encodes a receptor for glial cell-line derived neurotrophic factor (GDNF) on NCC and is crucial for ENS development (3-6). Loss-of-function mutations in RET account for up to 50% of familial cases and between 7-35% of sporadic cases of HSCR indicating that RET is the major HSCR gene (7-9). Other genes implicated in this disease that account for 7% of HSCR cases encode proteins involved in signaling pathways such as the endothelin 3/endothelin receptor B, and transcription factors SOX10, PHOX2B, TITF-1 which govern ENS development (1,10,11). SOX10 and TITF-1 bind to the RET promoters and induce the transcription from the RET promoter (10,12). Among the molecular regulators of gut development are the homeobox (HOX) genes, which consist of four gene clusters (A, B, C, D) encoding a family of transcription factors (13). In mammals, a total of 39 Hox genes located in 4 chromosomal clusters are organized in 13 paralogous groups. Some of the Hox genes of paralogous groups 4 and 5 are expressed in embryonic gut suggesting that these genes might form the Hox code for enteric development (14,15). In particular, Hoxb5 displays a distinct pattern of expression in mouse and human embryonic gut, suggesting a unique role of Hoxb5 in ENS and gut musculature development (14-17). Homozygous Hoxb5 knockout mice showed homeotic transformation of the skeleton but no abnormality of the ENS was reported (18). The lack of an ENS defect could be due to functional redundancy among Hox proteins. To investigate Hoxb5 function in ENS development and to circumvent the problem of functional redundancy, we generated transgenic mice that can be induced to express a chimaeric protein (enb5) in the NCC in mice by Cre/loxP. In this, the C-terminal DNA-binding homeodomain of Hoxb5 is linked to the N-terminal repressor domain of the Drosophila engrailed protein which has been shown to confer repressor activity when linked with heterologous DNA-binding domains. This chimeric enb5 repressor competes with wild-type Hoxb5 for binding to target genes and inhibits transcription, thereby blocking the developmental pathways that normally require Hoxb5. Our findings showed that NCC expressing enb5 failed to migrate to the distal intestine. We observed hypoganglionosis and slow peristalsis, and occasionally aganglionosis and intestinal obstruction in enb5-expressing mice (enb5/Cre). Ret expression was markedly reduced or absent in NCC and ganglia. More importantly, we demonstrated Hoxb5 trans-activated RET expression, which was abolished by enb5 (19) (Figures of the ENS phenotypes of enb5-expressing mice (enb5/Cre) and Hoxb5 transactivation of Ret are shown as Fig. 1 & 2 as attachment 1). Our data indicate Ret is a downstream target of Hoxb5 whose perturbation causes Ret haploinsufficiency, impairs NCC migration and leads to ENS phenotypes. The ENS defects in enb5-expressing mice resemble those in HSCR, suggesting the human HOXB5 gene may be mutated in this disease and/or defects in the activation of RET by HOXB5 could lead to RET haploinsufficiency and HSCR. We screened HOXB5 exons and those conserved non-coding sequences (CNS) flanking HOXB5 with significant homology between human and mouse for mutations or variations. In total, 188 HSCR patients and 189 healthy individuals of Chinese origin were screened, and three SNPs in the 3’ untranslated (UTR) region of the HOXB5 gene were found to be differentially represented (19). It is not know if these SNPs are functional or are markers in linkage disequilibrium with SNPs at the regulatory region implicated in HOXB5 expression. Regulatory elements for Hoxb5 expression in developing mouse have been identified at the 3’ end of HOXB5 (20). From the enb5 mouse data, it is tempting to speculate that a reduced amount of HOXB5 due to DNA variations in control regions could modulate RET expression and consequently the HSCR phenotype. By applying the HapMap data on Chinese Han from Beijing (CHB) and Sequenom technology for tag-SNPs distributed along the HOX clusters, genetic interactions were found among HOXA and HOXB loci and the RET loci tested, suggesting the interacting HOX loci may affect the penetrance of the RET-risk allele (21,22). Taken together all the data from us and others suggest that genes implicated in the signaling pathways governing ENS development may ultimately alter RET expression leading to HSCR. The penetrance of HSCR could be affected by combinations of mutations and/or DNA alterations (SNPs) in RET and in other HSCR genes (23,24) or other yet unidentified modifier loci (25,26). HSCR-associated SNPs in RET regulatory regions have been shown to decrease RET expression (10,21,27,28). Therefore, cis- and/or trans-regulatory defects in the activation of RET gene by transcription factors could lead to RET haploinsufficiency and HSCR disease. Our findings in mouse indicated that HOXB5 may represent a novel HSCR gene. Problems being addressed: (1) What are the roles of SNPs in the control regions implicated in the expression of HOXB5 gene (2) What are the roles of SNPs in the control regions of HOXB5 gene in the etiology of HSCR?

Project Title:	HOXB5 and RET promoter: regulation of RET transcription in Hirschsprungs disease
Investigator(s):	Lui VCH, Garcia-Barcelo MM, Tam PKH
Department:	Surgery
Source(s) of Funding:	General Research Fund (GRF)
Start Date:	10/2008

Abstract:

(1) To elucidate the mechanisms by which HOXB5 trans-activate RET; (2) To evaluate HOXB5 as a novel Hirschsprung’s disease locus.

Project Title:	Investigation of role of Hoxb5 in neural crest cell induction and maintenance in mouse embryos
Investigator(s):	Lui VCH, Cheung MCH
Department:	Surgery
Source(s) of Funding:	Seed Funding Programme for Basic Research
Start Date:	04/2009

Abstract:

Key Issues Neural crest cells (NCCs) are multipotent stem cells generated in the dorsal neural tube along the entire anterior posterior (A-P) axis of the neural tube. NCC progenitors initiate a distinct program of gene expression, undergo an epithelium to mesenchyme transition that will confer them ability to migrate. NCCs migrate throughout the body, where they differentiate into many different cell types including neurons and glia, cartilage, and melanoblasts (pigment cells). The generation of NCC progenitors and their lineage determinations are complex multistep process involving many genes. Abnormal NCC development give rises to a number of malformations collectively known as neurocristopathies, that affects many tissues including the nervous systems, skin pigmentation, skeletal structures, heart, dentures, and endocrine organs. Homeobox (Hox) genes, a group of highly conserved developmental control genes, are crucial in embryo patterning. In mammals, a total of 39 Hox genes located in 4 chromosomal clusters (Hoxa, Hoxb, Hoxc and Hoxd) are organized in 13 paralogous groups based on the structural and sequence homology. The dynamic expression patterns of Hox genes along the A-P axis of the neural tube are crucial in patterning the body axis and neuronal differentiation in the hindbrain. Hox genes also display dynamic patterns of expression along the dorsal-ventral (D-V) axis of the neural tube that correlate with the formation of different types of neurons. Abnormal D-V patterning of the neural tube may not only affect the formation of neurons, but could also influence the formation of NCC at the dorsal neural tube. Very little is known about the functions of Hox genes in the D-V patterning of the neural tube. Hoxb5 is expressed at low level at E8.5 in the presumptive hindbrain. At E12.5, Hoxb5 expression extends from the hindbrain throughout the entire neural tube with dorsally-restricted expression (1-3). The vagal NCCs that emigrate from the neural tube at the level of somite 1-7 also express Hoxb5 migrate to the developing gut and form the enteric nervous system (ENS). To investigate Hoxb5 function in vagal NCCs and to circumvent the problem of functional redundancy, we generated transgenic mice that can be induced by Cre recombinase to express a dominant negative chimaeric protein (enb5) containing the mouse Hoxb5 homeodomain fused to the transcription repressor of Drosophila engrailed repressor. Enb5 is expected to function as a dominant transcriptional repressor to compete with the endogenous Hoxb5 for binding to the regulatory elements of its downstream target genes and repress their transcription thereby inactivating Hoxb5 functions. By crossing with transgenic mice that express Cre recombinase specifically in vagal NCCs, we previously showed that perturbation of Hoxb5 function caused defective vagal NCC migration in the developing intestine (4). To investigate the functions of Hoxb5 in nervous system development, we crossed our enb5 transgenic mice to Wnt1Cre mice. Wnt1 regulatory elements direct the expression of Cre to the dorsal neural tube, NCC progenitors, the midbrain and the cerebellum in Wnt1Cre mice (5,6). Wnt1Cre/enb5 double transgenic mice display hydrocephalus, enlarged and domed craniae, abnormal skin pigmentation, and ENS defects. In this proposal, we shall investigate the molecular mechanisms by which skin pigmentation is disrupted in Wnt1Cre/enb5 mice. Melanoblasts differentiate from the NCC and migrate along the dorsolateral pathway between the dermatome and the epidermis giving rises to all the pigment cells of the skin. The skin pigmentation defects were first noticeable in Wnt1Cre/enb5 mice at postnatal day 3 (Fig.1A), and white hair was clearly seen at the time of weaning at the trunk (Fig. 1B). To monitor the localization of Wnt1Cre expressing cells, Wnt1Cre mice were crossed with Cre reporter mouse strain Rosa26-Cre reporter mouse (R26R) (7) to generate Wnt1Cre/R26R mice, which were then crossed with enb5 mice to obtain Wnt1Cre/enb5/R26R mutant and Wnt1Cre/R26R control mice. X-gal staining and sectioning of adult skin revealed no melanocytes at the unpigmented skin of Wnt1Cre/enb5/R26R mouse (Fig. 1C). Melanoblasts were found residing in the dermis and also penetrating into the epidermis of Wnt1Cre/R26R E12.5 embryos. In contrast, melanoblasts were undetectable in Wnt1Cre/enb5/R26R embryos (Fig. 2). The number of NCC progenitors was drastically reduced at the dorsal neural tube of Wnt1Cre/enb5/R26R embryos as compared to that of Wnt1Cre/R26R mice. Our data suggested that early induction and/or maintenance of NCC progenitors may be impaired leading to a reduction of melanoblasts, and hypopigmentation in Wnt1Cre/enb5 mice. Problems being addressed We shall determine the molecular mechanisms by which (i) perturbation of Hoxb5 functions in the neural tube causes the reduction of NCC in Wnt1Cre/enb5 embryos, and (ii) Hoxb5 induces and/or maintains the NCC progenitors pool in early embryos. References :(PI is highlighted with *): 1. Holland, P. W., and Hogan, B. L. (1988) Spatially restricted patterns of expression of the homeobox-containing gene Hox 2.1. during mouse embryogenesis. Development 102, 159-174 2. Krumlauf, R., Holland, P. W., McVey, J. H., and Hogan, B. L. (1987) Developmental and spatial patterns of expression of the mouse homeobox gene, Hox 2.1. Development 99, 603-617 3. Kuratani, S. C., and Wall, N. A. (1992) Expression of Hox 2.1 protein in restricted populations of neural crest cells and pharyngeal ectoderm. Dev Dyn 195, 15-28 4. Lui, V. C*., Cheng, W. W., Leon, T. Y., Lau, D. K., Garcia-Barcelo, M. M., Miao, X. P., Kam, M. K., So, M. T., Chen, Y., Wall, N. A., Sham, M. H., and Tam, P. K. (2008) Perturbation of hoxb5 signaling in vagal neural crests down-regulates ret leading to intestinal hypoganglionosis in mice. Gastroenterology 134, 1104-1115 5. Chai, Y., Jiang, X., Ito, Y., Bringas, P., Jr., Han, J., Rowitch, D. H., Soriano, P., McMahon, A. P., and Sucov, H. M. (2000) Fate of the mammalian cranial neural crest during tooth and mandibular morphogenesis. Development 127, 1671-1679 6. Danielian, P. S., Muccino, D., Rowitch, D. H., Michael, S. K., and McMahon, A. P. (1998) Modification of gene activity in mouse embryos in utero by a tamoxifen-inducible form of Cre recombinase. Curr Biol 8, 1323-1326 7. Soriano, P. (1999) Generalized lacZ expression with the ROSA26 Cre reporter strain. Nature genetics 21, 70-71

Project Title:	Wif-1 regulates cloaca septation via Wnt signaling in urorectal development
Investigator(s):	Lui VCH
Department:	Surgery
Source(s) of Funding:	Seed Funding Programme for Basic Research
Start Date:	03/2010

Abstract:

Objectives of the research proposal Key Issues Urorectal development includes 3 distinct processes: outgrowth of the genital tubercle (GT), cloaca septation and urethra tubularization (sex differentiation). Cloaca is the primitive outlet of the urinary and intestinal stream, locating at the caudal extreme end of the embryo. It is an endoderm-lined hollow region covered by a two to three-cell-thick membrane, known as cloaca membrane. A sheet of mesenchyme called urorectal septum (urs) originated from the cranial cloaca grows and descends to separate the hindgut (intestinal stream) from the urethra (urinary stream), and eventually reaches the cloaca membrane. This process is termed as cloaca septation. Cloaca septation is a complex multistep process requiring lateral-to-medial and cranial-to-caudal growth of endodermally lined mesenchyme that involves many genes (Dravis, Yokoyama et al. 2004). Failure of cloaca septation leads to anorectal malformations (ARMs) or hypospadias. The urs descent and cloaca septation initiate and complete between E12.5 and E13.5 in mice, and between E14.5 and E15.5 in rats. Wnt signaling pathway controls diverse developmental processes including palate, limbs, kidneys and CNS. There are 19 Wnt ligands and several signaling pathway inhibitors such as Dickkopf, Wnt inhibitory factor-1 (Wif-1), sFRP and cerberus. Wnt signaling polymerase chain reaction array analysis and immunohistochemistry indicate that Wnt ligands and Wif-1 are expressed in the developing mouse and rat urorectal region (Fig. 1, 2 and data not shown. See Attachment). Wnt5a is expressed at the developing GT mesenchyme, and it modulates the outgrowth of GT. The interaction between Wnt and Sonic Hedgehog (Shh) signaling is indispensable in the development of many tissues including papilla, neural tube, and midbrain floor plate. Wnt and Shh have also been implicated in urorectal development at the initial GT outgrowth and the late androgen-dependent tubularization. Sonic Hedgehog (Shh), Wnt5a and Tcf1/Tcf4 (target gene of Wnt canonical signaling) homozygous knockout embryos displayed complete agenesis of the GT (Yamaguchi, Bradley et al. 1999; Haraguchi, Mo et al. 2001). Knocking out Hoxa13, a downstream target of Shh, reduced androgen signal and disturbed the closure of urethral groove in the ventral proximal GT leading to hypospadias (Morgan, Nguyen et al. 2003). Gli2 and Gli3 are the downstream mediators of Sonic Hedgehog (Shh) signaling. Gli2 and Gli3 mutant mice demonstrated different forms of ARMs (Kimmel, Mo et al. 2000; Mo, Kim et al. 2001). However, there is lack of knowledge about genetic programs governing cloaca septation, the descent of urs separating urogenital sinus and rectum. To investigate the molecular changes associated with cloaca septation and the possibilities of the associations between the urs descent and hypospadias as well as ARMs, Affymetrix GeneChip analysis was performed to identify differentially expressed genes between ETU-treated and normal rat embryos at E14.5. ETU is a well-established drug to induce ARMs in rat fetuses by intragastric administration to pregnant rats. Array analysis and subsequent confirmation by real-time RT-PCR in biological replicates revealed up-regulation of Wnt inhibitory factor-1 (Wif-1) in ETU-treated embryos (Fig. 2 and data not shown. See Attachment). ETU-treated embryos displayed urorectal developmental defects typified in human hypospadisis and ARMs including delayed genital development, untubularized penile urethra, and the presence of internal fistula of vagina to rectum and urethra. Immuno-histochemical analysis on E14.5 to E16.5 rat embryos revealed that Wif-1 was highly expressed at the ventral cloaca canal, ventral urethral endoderm and anal opening. The spatiotemporal distribution of Wif-1 protein at the cloaca epithelium was disrupted in ETU-treated rats (Fig.2). Taken together all these data suggest that Wif1 is spatiotemporally expressed around the cloaca epithelium revealing a presumable involvement in the descent of urs. We hypothesize that disturbance of Wif1 expression in the urorectal septum could result in defective urorectal development. In this proposal, we aim to investigate the functional roles of Wif-1, Wnt and Shh signaling pathways in cloaca septation, in particular, the descent of urs separating urogenital sinus and hindgut using in vitro organotypic culture and mutant mice. Problems being addressed We shall investigate the molecular mechanisms of urorectal development in which (i) Dorsal-ventral differential expression of Wif-1 governs the process of cloaca septation, (ii) Wnt and Shh signaling pathways coordinately regulate urorectal development. References: Dravis, C., N. Yokoyama, et al. (2004). "Bidirectional signaling mediated by ephrin-B2 and EphB2 controls urorectal development." Dev Biol 271(2): 272-90. Haraguchi, R., R. Mo, et al. (2001). "Unique functions of Sonic hedgehog signaling during external genitalia development." Development 128(21): 4241-50. Kimmel, S. G., R. Mo, et al. (2000). "New mouse models of congenital anorectal malformations." J Pediatr Surg 35(2): 227-30; discussion 230-1. Mo, R., J. H. Kim, et al. (2001). "Anorectal malformations caused by defects in sonic hedgehog signaling." Am J Pathol 159(2): 765-74. Morgan, E. A., S. B. Nguyen, et al. (2003). "Loss of Bmp7 and Fgf8 signaling in Hoxa13-mutant mice causes hypospadia." Development 130(14): 3095-109. Yamaguchi, T. P., A. Bradley, et al. (1999). "A Wnt5a pathway underlies outgrowth of multiple structures in the vertebrate embryo." Development 126(6): 1211-23.

List of Research Outputs

Zhang M., Lui V.C.H., Tam P.K.H. and Sham M.H., Abnormal Enteric Nervous System Development in a Sox10^NGFP Mouse Mutant, 14th Research Postgraduate Symposium, December 2 & 3, 2009, The University of Hong Kong. 2009.

Researcher : Lui WT

List of Research Outputs

Lui W.T. and Wong N.S., Functional significance of the AU-rich sequence element in the mRNA of the stree-inducible protein CHOP/Gadd153, 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium, CUHK, Hong Kong, 15 May,. 2010.

Researcher : Mak ACY

List of Research Outputs

Mak A.C.Y., Bioinformatic and Functional Approaches to Identify Potential SOX9 Target Genes in Inner Ear Development, PhD Thesis, 2010.

Mak A.C.Y., Szeto Y.Y., Fritzsch B. and Cheah K.S.E., Differential and overlapping expression pattern of SOX2 and SOX9 in inner ear development, Gene Expression Patterns. 2009, 9: 444-453.

Researcher : Mak CYA

List of Research Outputs

Mak C.Y.A., Bioinformatic and functional approaches to identify genes regulated by Sox9 in inner ear development, June 2010, PhD Thesis. 2010.

Researcher : Mak TY

List of Research Outputs

Chan C.P., Mak T.Y., Chin K.T. and Ng I.O.L., N-linked glycosylation is required for optimal proteolytic activation of membrance-bound transcription factor CREB-H, 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium, CUHK, Hong Kong, 15 May. 2010.

Chan C.P., Mak T.Y., Chin K.T., Ng I.O.L. and Jin D., N-linked glycosylation is required for optimal proteolytic activation of membrane-bound transcription factor CREB-H., J. Cell Sci.. The Company of Biologists, 2010, 123: 1438-1448.

Chan C.P., Mak T.Y., Chin K.T. and Jin D., Requirement of N-linked Glycosylation in the Luminal Domain for Optimal Proteolytic Activation of Liver-enriched transcription Factor CREB-H, 14th Research Postgraduate Symposium, December 2 & 3, 2009, The University of Hong Kong. 2009.

Mak T.Y., Chan C.P. and Jin D., Differential stability and transcriptional activity of CREB3 subfamily transcription factors, 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium, CUHK, Hong Kong, 15 May,. 2010.

Researcher : Mak TY

List of Research Outputs

Researcher : Melhado IG

List of Research Outputs

Cheah K.S.E., Wang C., Tan Z., Melhado I.G., Niu B., Tsang K.Y., Zhang M...Q..., Ron D... and Chan D., Alleviating ER stress in chondrocytes: survival strategies with double-edged developmental consequences , 43rd Annual Meeting for the Japanese Society of Developmental Biologists Jointly Sponsored by the Asia-Pacific Developmental Biology Network, Kyoto, Japan, 20 June 2010.

Cheah K.S.E., Wang C., Tan Z., Melhado I.G., Niu B., Tsang K.Y., Zhang M...Q..., Ron D. and Chan D., Genetic and genomic analyses of chondrocyte adaptation to ER stress, The Genetics Society Spring Meeting “Mouse Genetics – Think Globally, Act Locally” The Wellcome Trust Conference Centre, Hinxton, UK, 23 April 2010 . 2010.

Leung Y.L., Gao B., Melhado I.G., Leung K.K.H., Wynn S.L., Chan D. and Cheah K.S.E., Sox9 mediates gene expression specificity in growth plate chondrocytes via concomitant positive and negative regulation, 55th Annual Meeting of the Orthopaedic Research Society, New Orleans, USA. 2010.

Tan Z., Niu B., Tsang K.Y., Melhado I.G., Zhang M., Chan D. and Cheah K.S.E., Global gene expression changes during chondrocyte adaptation to ER stress, 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium, CUHK, Hong Kong, 15 May,. 2010.

Researcher : Miao Y

List of Research Outputs

Miao Y., In: Development of a Bioinformatics and Statistical Framework to Integrate Biological resources for Genome-wide Genetic Mapping and Its Applications, PhD Thesis. 2009.

Miao Y., The Localization of E. coli Persistent Gene Products, PhD Thesis, 2010.

Miao Y., Danchin A.L.M. and Huang J., Visualizing the Proteome Escherichia coli by Recombineering Technology, 14th Research Postgraduate Symposium, December 2 & 3, 2009, The University of Hong Kong. 2009.

Researcher : Miao Y

List of Research Outputs

Miao Y., In: Development of a Bioinformatics and Statistical Framework to Integrate Biological resources for Genome-wide Genetic Mapping and Its Applications, PhD Thesis. 2009.

Miao Y., The Localization of E. coli Persistent Gene Products, PhD Thesis, 2010.

Researcher : Ng KT

List of Research Outputs

Cheung M.C.H., Chau K.H.B., Chan A.Y.L., Ng K.T., Wu M.H. and Briscoe J.A.M.E.S., Regulation of Sox9 Function by SUMOylation in avian neural crest development, In: Shinichi Aizawa Maria Leptin Nancy Papalopulu Claudio D Stern Dider Stainer Patrick Tam, Mechanisms of Development 16th International Society of Developmental Biologists Congress 2009 Book of Abstracts. Elsevier, 2009, 126: S308.

Researcher : Ng MH

List of Research Outputs

Ng M.H., Ng R.K., Kong C.T., Jin D. and Chan L.C., Activation of Ras-dependent Elk-1 activity by MLL-AF4 family fusion oncoproteins, Experimental Hematology. 2010, 38: 481-488.

Ng M.H., American Society for Virology Student Travel Award, American Society for Virology. 2009.

Ng M.H., Siu K.L., Kok K.H., Ching Y.P. and Jin D., MIP-T3 is a novel inhibitor in TBK1/IRF3-dependent antiviral response. , 28th Annual meeting of the American Society for Virology. 2009.

Ng M.H., Siu K.L., Ching Y.P. and Jin D., MIP-T3 is a novel inhibitor in TBK1/IRF3-dependent antiviral response, The American Society for Virology 28th Annual Meeting, University of British Columbia, Vancouver, BC, Canada. 2009.

Ng M.H., Negative Regulation Of Type-I Interferon Production By MIP-T3, PhD Thesis. 2009.

Ng M.H., Travel Award, American Society for Virology. 2009.

Researcher : Ng MH

List of Research Outputs

Ng M.H., Ng R.K., Kong C.T., Jin D. and Chan L.C., Activation of Ras-dependent Elk-1 activity by MLL-AF4 family fusion oncoproteins, Experimental Hematology. 2010, 38: 481-488.

Ng M.H., American Society for Virology Student Travel Award, American Society for Virology. 2009.

Ng M.H., Siu K.L., Kok K.H., Ching Y.P. and Jin D., MIP-T3 is a novel inhibitor in TBK1/IRF3-dependent antiviral response. , 28th Annual meeting of the American Society for Virology. 2009.

Ng M.H., Negative Regulation Of Type-I Interferon Production By MIP-T3, PhD Thesis. 2009.

Ng M.H., Travel Award, American Society for Virology. 2009.

Researcher : Ni Y

List of Research Outputs

Lin R., Lau C.F., Wang J., Ni Y., Fung M.L. and Huang J., Reduced neuronal nitric oxide production in KIF5b-deficiency mice, 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium, CUHK, Hong Kong, 15 May,. 2010.

Researcher : Niu B

Project Title:	Whole genome identification of active core promoters, enhancers and insulators in mouse growth plate
Investigator(s):	Niu B, Cheah KSE
Department:	Biochemistry
Source(s) of Funding:	Small Project Funding
Start Date:	01/2010

Abstract:

Transcription initiation is the key step in the regulation of gene expression. During this process, transcription factors bind promoter region of a gene in a sequence specific manner and recruit RNA polymerase II to form an active initiation complex around the transcription start site (TSS). There are typically 3 types of promoters: (1) a core promoter that is ~100 bp long around the TSS containing binding sites for RNA polymerase II (Pol II) and general transcription factors; (2) a proximal promoter of several hundred base pairs long containing primary specific regulatory elements located immediately upstream of the core promoter; and (3) a distal promoter up to thousands of base pairs long providing additional regulatory information. As it contains primary information to control gene transcription, it is a fundamental step to identify the core-promoter in study of gene expression patterns and constructing gene transcription networks. In the past 10 yrs, dozens of computational methods have been developed to predict core promoters and enhancers [1]. However, it is still a challenging problem to identify functional TSS accurately at high resolution, especially for the non-CpG-related promoters. Up to now, almost all of the existing core-promoter prediction methods are based on DNA sequence features, few of them has incorporated the epigenetic information. Eukaryotic DNA is packaged into chromatin structure. The basic unit of chromatin is the nucleosome that is composed of an octamer of four core histones (H2A, H2B, H3, and H4) wrapped by 147-bp-long DNA. The modifications of the histones are found to be associated with transcription initiation and elongation. Recently, taking advantage of next-generation sequencing technology, researchers developed the chromatin immunoprecipitation followed by sequencing (ChIP-seq) technology and obtained high-throughput genome-wide profiles of histone modification patterns in mammalian cells [2]-[7]. These high-resolution histone modification profiling data shed new light on studying regulation mechanism of gene transcription. Lander and colleagues [2] identified over 1000 large intergenic noncoding RNA (lincRNA) genes in mouse ES, MEF and neural progenitor cells by analyzing the DNA regions enriched for H3K4 and H3K36 tri-methylation. These lincRNA genes are neighboring to those genes encoding transcription factors along DNA, and are bound by the transcription factors Sox2, Oct4 and Nanog in ES cells, suggesting they may play important roles in cell self-renewal. Ren and colleagues [3]-[4] determined the chromatin modification states in high resolution in human HeLa cells. By using a computational algorithm capable of learning the patterns of histone methylation and acetylation, they identified 198 core promoters and 86 enhancers in a 30Mb genomic region. Zhao and colleagues [5]-[6] generated high-resolution maps for the genome-wide distribution of 39 histone modifications in human T cells. Typical patterns of histone methylation were observed at promoters, insulators, enhancers, and transcribed regions. They also found that the mono-methylations of H3K27, H3K9, H4K20, H3K79, and H2BK5 are linked to gene activation, whereas trimethylations of H3K27, H3K9, and H3K79 are linked to repression. A priority of the Area of Excellence (AoE) scheme in Development Genomics and Skeletal Research is to build up the strength in iPS and stem cell research for regenerative medicine, which holds the promise of leading to new cures and better individualised treatment to skeletal disorders. iPS cells, of particular interest, can be taken from adults and reprogrammed to an embryonic state, from which they can develop into chrondrocyte, osteoblast, osteoclasts, and mesenchymal stem cells to regenerate new skeletal tissues. However, it is still not clear whether iPS can perform as well as truly embryonic cells in reprogramming and differentiation. By mapping and comparing the epigenetic profiles, we are able to gain more insights into the nature of these cells. Under AoE, a central topic is to investigate the properties of chrondrocyte in mouse growth plate development. We have performed microArray experiments by using the samples collected from the mice growth plates that are cryosectioned over the developmental stages. We observed that the expression levels of several hundreds of genes have been changed during proliferation, differentiation and apoptosis. To elucidate the epigenetic mechanism underlying these processes, we propose to: i) perform ChIP-sequencing experiments to collect the signatures of H3K4me2, H3K4me3, H3K27me3 and H3K36me3 in whole genome; ii) develop computational algorithm to analyze the patterns of the signatures; iii) identify new core promoters, enhancers, and insulators functional for development by leveraging the patterns learned; iv) create the gene network that incorporate the identified elements to find out the core regulatory circuitry. In pilot study, an algorithm has been developed to predict core promoters by using the H3K4me3 and the H3K36me3 signatures. Result of 5-fold cross-validation indicates that our method is more accurate and efficient than the one used by Ren et al. [3]. But there still more rooms for improvements.

List of Research Outputs

Cheah K.S.E., Wang C., Tan Z., Melhado I.G., Niu B., Tsang K.Y., Zhang M...Q..., Ron D... and Chan D., Alleviating ER stress in chondrocytes: survival strategies with double-edged developmental consequences , 43rd Annual Meeting for the Japanese Society of Developmental Biologists Jointly Sponsored by the Asia-Pacific Developmental Biology Network, Kyoto, Japan, 20 June 2010.

Cheah K.S.E., Wang C., Tan Z., Melhado I.G., Niu B., Tsang K.Y., Zhang M...Q..., Ron D. and Chan D., Genetic and genomic analyses of chondrocyte adaptation to ER stress, The Genetics Society Spring Meeting “Mouse Genetics – Think Globally, Act Locally” The Wellcome Trust Conference Centre, Hinxton, UK, 23 April 2010 . 2010.

Tan Z., Niu B., Tsang K.Y., Melhado I.G., Zhang M., Chan D. and Cheah K.S.E., Global gene expression changes during chondrocyte adaptation to ER stress, 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium, CUHK, Hong Kong, 15 May,. 2010.

Tan Z., Niu B., Chan D. and Cheah K.S.E., Mechanisms Underlying Chondrocyte Reprogramming Surviving ER Stress, 14th Research Postgraduate Symposium, December 2 & 3, 2009, The University of Hong Kong. 2009.

Researcher : Niu G

List of Research Outputs

Liu Z., Liu M., Niu G., Cheng Y. and Fei J., Genome-wide identification of target genes repressed by the zinc finger transcription factor REST/NRSF in the HEK 293 cell line., Acta Biochim Biophys Sin (Shanghai). 2009, 41: 1008.

Yao M., Niu G., Sheng Z., Wang Z. and Fei J., Identification of a Smad4/YY1-recognized and BMP2-responsive transcriptional regulatory module in the promoter of mouse GABA transporter subtype I (Gat1) gene., J Neurosci. 2010, 30: 4062.

Researcher : Qi S

List of Research Outputs

Qi S., Sun H. and Huang J., Expression and Purification of Mutated hpn Protein, and Studies of Function of Cystein Residues in hpn, 14th Research Postgraduate Symposium, December 2 & 3, 2009, The University of Hong Kong. 2009.

Qi S., Sun H. and Huang J., Expression and purification of mutated hpn protein and studies of cysteine residues in hpn, 14th International Conference on Biological Inorganic Chemistry, Nagoya, Japan, July 25-30, 2009 (published in J. Biol Inorg Chem. 2009, 14, Suppl1, P606). 2009.

Researcher : Qi S

List of Research Outputs

Researcher : Sam L

List of Research Outputs

Lui E.L.H., Shum K.T., Sam L., Wang Y., Watt R.M. and Tanner J.A., Aptamers Against Polyphosphate Kinase 2 (PPK2) From Mycobacterium Tuberculosis As A Potential Approach Of Novel Anti-Tuberculosis Therapy , CUHK Croucher Advanced Study Institute “Structure-Based Screening and Design of Ligands for Protein Targets” . 2009.

Researcher : Samartzis D

List of Research Outputs

Li J., Cheung K.M.C., Samartzis D., Zhu X.D., Li M. and Luk K.D.K., Key-Vertebral Screw Strategy (KVSS): an alternative surgical strategy for main thoracic curve correction in patients with adolescent idiopathic scoliosis, 16th International Meeting on Advanced Spine Techniques, Vienna, Austria, July 15-18, 2009 . 2009.

Samartzis D., Karppinen J., Mok P.S., Fong D.Y.T., Luk K.D.K. and Cheung K.M.C., Skipped level disc degeneration of the lumbar spine, 2010 Annual Meeting of the American Academy of Orthopaedic Surgeons, New Orleans, Louisiana, March 9-13, 2010.

Researcher : Sham MH

Project Title:	International Congress on Differentation and Cell Biology Neural-specific Cis-acting and Trans-acting Regulation of the Mouse Hoxb-3 Gene During Embryogenesis
Investigator(s):	Sham MH
Department:	Biochemistry
Source(s) of Funding:	URC/CRCG - Conference Grants for Teaching Staff
Start Date:	09/2000

Abstract:

N/A

Project Title:	*Genetic and developmental defects of a mouse mutant Mcc* with microphthalmia, cataract and closed eyelid**
Investigator(s):	Sham MH, Song Y
Department:	Biochemistry
Source(s) of Funding:	General Research Fund (GRF)
Start Date:	09/2006
Completion Date:	02/2010

Abstract:

To identify the Mcc mutant gene locus by fine mapping and to determine the mutation which cause the abnormal eye phenotypes; to characterize the developmental defects of the Mcc mutant eye and lens during embryonic, postnatal and adult stages at the cellular and molecular levels, in order to correlate the genotype with the phenotype.

Project Title:	Genetic interaction of Sox10 and Gli3 in mouse inner ear development
Investigator(s):	Sham MH
Department:	Biochemistry
Source(s) of Funding:	Seed Funding Programme for Basic Research
Start Date:	04/2009
Completion Date:	03/2010

Abstract:

The Sox10 gene encodes a conserved HMG-domain DNA binding transcription factor which is expressed in neural crest cells and a variety of neural crest derivatives during mammalian development. Defects in neural crest development lead to congenital birth defects in humans. SOX10 mutations underlie the neurocristopathies Waardenburg–Hirschsprung and Yemenite deaf-blind hypopigmentation. Waardenburg-Shah syndrome (WS) individuals with SOX10 heterozygous mutations exhibit sensorineural deafness and pigmentation defects, as well as intestinal obstruction. Sox10 is expressed in the otic vesicle from early stages, and is proposed to regulate differentiation and patterning of the otic vesicle. It has been suggested that the sensorineural deafness and pigmentation defects in some WS patients are caused by a deficiency of melanocytes in the skin and stria vascularis of the inner ear. It has also been postulated that the absence of melanocytes in the inner ear would result in the loss of endolymphatic potential and cause endolymphatic collapse and sensory hair cell degeneration in the organ of Corti. However, the expression and function of Sox10 in the stria vascularis has not been clearly demonstrated experimentally. In our laboratory we have generated a Sox10 mutant in which the EGFP reporter gene is knocked-in to the Sox10 locus. From our preliminary study of Sox10 mouse mutant phenotype, abnormal neural innervation to the sensory hair cells may also contribute to hearing loss. Therefore, how SOX10 mutations lead to deafness in WS patients remain unknown to date. In addition to factors expressed in the otic vesicle, development of the mammalian inner ear is dependent on signals from the surrounding tissues. Sonic hedgehog (Shh) secreted from the floor plate and notochord is required for specification of inner ear structures. The Gli transcription factors expressed in the otic epithelium are required to mediate Shh signaling. It has been shown that Gli3 repressor is required for pattering dorsal inner ear structures, while Gli activator proteins are essential for ventral inner ear structures. Recently, in a mutagenesis screening experiment, it has been shown that Gli3 is a modifier of Sox10 function, the hypopigmentation defect exhibited by Sox10 haploinsufficient mutant mice become more severe when Gli3 is also mutated. Therefore, it will be interesting to elucidate whether Gli3 also serves as a genetic modifier for the function of Sox10 in inner ear development, hence contributing to the varied severities of sensorineural deafness displayed by Waardenburg-Shah syndrome patients. The aim of this project is to elucidate the functions of Sox10 during inner ear development and to understand the molecular basis for hearing loss caused by Sox10 mutation. The specific objectives of this project are: 1. To investigate the inner ear defects of the Sox10N-GFP mouse mutant to examine potential melanocyte deficiency in the stria vascularis and neural innervation defects in the mutant. 2. To investigate the genetic interaction of Gli3 and Sox10 in inner ear development by analyzing the inner ear phenotypes Gli3;Sox10N-GFP double mutant mice.

Project Title:	Sonic hedgehog signaling and cross talks with Hox genes in hindbrain neurogenesis
Investigator(s):	Sham MH
Department:	Biochemistry
Source(s) of Funding:	General Research Fund (GRF)
Start Date:	10/2009

Abstract:

1) To investigate the regulation of the Nkx2.2 gene by Hoxb1 and Gli by biochemical studies and genetic analysis in order to reveal how Hox and Gli genes interact to integrate AP and DV patterning; 2) To dissect the Shh signaling pathway by knockout of Shh signal transducers in mice in order to identify the intracellular signaling requirement for mediating Shh pathway in rhombomere 4 of the hindbrain.

Project Title:	Outstanding Research Student Supervisor Award 2008-2009
Investigator(s):	Sham MH
Department:	Biochemistry
Source(s) of Funding:	Outstanding Research Student Supervisor Award
Start Date:	12/2009

Abstract:

For recognizing, rewarding and encouraging exceptioal research achievements; and for strengthening the research culture of the University.

Project Title:	The role of Irx3 and Irx5 homeobox genes in motor neurone specification
Investigator(s):	Sham MH
Department:	Biochemistry
Source(s) of Funding:	Seed Funding Programme for Basic Research
Start Date:	04/2010

Abstract:

The Iroquois homeobox (Irx) genes encode transcription factors of the TALE (three amino acid loop extension) class of homeodomain-containing proteins. In the mouse genome, there are six Irx genes which are organized into two clusters: the IrxA cluster contains Irx1, Irx2 and Irx4, and the IrxB cluster contains Irx3, Irx5 and Irx6. Vertebrate Irx genes are widely expressed in the central nervous system and they are suggested to have important patterning and specification functions during neural development. In chick, several Irx genes were identified as key players in patterning brain development. However, analysis of mutant mice has revealed that Irx2, Irx4 or Irx5 have only mild phenotypes, suggesting that these genes share overlapping developmental functions. Irx3 is expressed in a variety of tissues, especially in the developing central nervous system (CNS) including mesencephalon, metencephalon and neural tube. Irx3 was first identified as a Class I progenitor gene implicated in ventral neural tube patterning. The expression of Irx3 in the spinal cord is repressed by sonic hedgehog signalling; mutual-repressive interactions between Irx3 and the bHLH transcription factor Olig2 are thought to establish the boundary of progenitor populations for V2 and motor neurones. It is believed that the expression of Irx3 in the p2 progenitor domain; and its repressive activity in restricting the dorsal limit of the pMN domain prevents motor neurone generation. We have been interested in hindbrain neurogenesis and we have focussed our study on the facial branchial motoneurones originated from rhombomere(r) 4 of the developing hindbrain. We have shown that in hindbrain r4, Hoxb1 interact with Gli transcription factors to activate the expression of Nkx2.2 in the p3 progenitor domain in the ventral region. To understand the functions of Irx3 and Irx5 in hindbrain neurogenesis, we first examined the phenotypes of Irx3 and Irx5 knockout mutant mice, as well as Irx3/Irx5 double knockout mice in which the linked loci of both Irx3 and Irx5 are deleted by gene targeting. Using markers for dorsoventral neuronal patterning genes, we examined the expression of Nkx2.2 (which marks the p3 progenitor domain and branchial motoneurones) and Olig2 (which marks the p2 progenitor domain and somatic motoneurones) in different hindbrain rhombomeres. Interestingly, in homozygous Irx3/5 double knockout mutant embryos, the expression of Nkx2.2 was unaffected, but ectopic expression of Olig2 could be detected specifically in r4 but not in other rhombomeres (see Fig.1 in the Appendix). Therefore, in the absence of both Irx3 and Irx5 genes and their repressive functions, Olig2 is de-repressed and ectopically activated, but only in r4. In other rhombomeres of the hindbrain, other genes would be responsible for restricting the expression of Olig2. To further decipher whether the de-repression of Olig2 expression was due to the function of Irx3 or Irx5, we examined the expression of Nkx2.2 and Olig2 in the single mutant embryos. Interestingly, Olig2 positive cells could also be detected in r4 of Irx3-/- and Irx5-/- mutant embryos (Fig. 2). To investigate the identity of the ectopic Olig2 positive cells, we used a motor neurone marker MNR2 and observed that ectopic somatic motoneurones could be found only in the Irx3/5-/- double mutant embryos, but not in the single mutants (Fig. 2). Therefore, in the Irx3-/- or Irx5-/- mutants, ectopic Olig2 expression was insufficient to specify the formation of somatic motoneurones. This implies an important observation that both Irx3 and Irx5 are required in r4 to negatively regulate the expression of Olig2 and specifty the motoneurone subtype identity. Based on our results, we hypothesize that the Irx3 and Irx5 genes share functional redundancy. In addition to Irx3, Irx5 can also negatively regulate the expression of Olig2. The aim of this project is to elucidate the roles of the two genes Irx3 and Irx5 in the specification of motor neurone subtypes in the hindbrain and the spinal cord. We will address the following specific objectives: 1. To investigate the role of Irx5 in the regulation of Olig2 and neuronal subtype specification in the developing central nervous system including the hindbrain and spinal cord. 2. To investigate the functional roles of Irx3 and Irx5 in hindbrain rhombomere 4 neuronal specification by analysis of the phenotypes of Irx3/5 double mutant mice.

Project Title:	International Society for Stem Cell Research 8th Annual Meeting Hoxb1 cooperates with Gli to mediate sonic hedgehog signaling in hindbrain neural progenitor cell fate determination
Investigator(s):	Sham MH
Department:	Biochemistry
Source(s) of Funding:	URC/CRCG - Conference Grants for Teaching Staff
Start Date:	06/2010
Completion Date:	06/2010

Abstract:

N/A

List of Research Outputs

Au Yeung K.L., Sze K.Y., Sham M.H. and Chan B.P., Development of a micromanipulator-based loading device for mechanoregulation study of human mesenchymal stem cells in three-dimensional cellagen constructs , Tissue Engineering, Part C Methods. 2010, 16(1): 93-107.

Au-yeung K.L., Sze K.Y., Sham M.H. and Chan B.P., Development of a micromanipulator-based loading device for mechanoregulation study of human mesenchymal stem cells in 3D collagen constructs., WACBE (World Association for Chinese Biomedical Engineers) World Congress on Bioengineering 2009. 26-29 Jul 2009, Hong Kong, China.. 2009, 53.

Chan K.T., Qi J. and Sham M.H., Multiple coding and non-coding RNAs in the Hoxb3 locus and their spatial expression patterns during mouse embryogenesis, Biochem Biophys Res Commun. 2010, 23:398(2): 153-9.

Chan S.C.L. and Sham M.H., Hox Expression Profile and its Epigenetic Regulation during Chondrogenesis, 14th Research Postgraduate Symposium, December 2 & 3, 2009, The University of Hong Kong. 2009.

Chu K.H., Zhang M., Wong E.Y.M., Szeto Y.Y., Chan Y.S., Cheah K.S.E. and Sham M.H., Best Poster Award: Sox10 mutation affects cochleo-vestibular ganglion gliogenesis, 43rd Annual Meeting for the Japanese Society of Developmental Biologists, Kyoto, Japan, 20-23 June, 2010.. 2010.

Chu K.H., Zhang M., Wong E.Y.M., Szeto Y.Y., Chan Y.S., Cheah K.S.E. and Sham M.H., Best Poster Award: Sox10 mutation affects gliogenesis of the cochleo-vestibular ganglion, 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium, 15 May 2010. 2010.

Chu K.H., Zhang M., Wong E.Y.M., Szeto Y.Y., Chan Y.S., Cheah K.S.E. and Sham M.H., Sox10 mutation affects cochleo-vestibular ganglion gliogenesis, 43rd Annual Meeting for the Japanese Society of Developmental Biologists, Kyoto, Japan, 20-23 June. 2010.

Chu K.H., Zhang M., Wong E.Y.M., Szeto Y.Y., Chan Y.S., Cheah K.S.E. and Sham M.H., Sox10 mutation affects gliogenesis of the cochleo-vestibular ganglion, 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium, 15 May 2010.

Lei I.L., Gao X.L., Wang Z. and Sham M.H., BAF250a-containing complex is required for secondary heart field development, 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium, CUHK, Hong Kong, 15 May,. 2010.

Ma L., Zheng L., Sham M.H. and Cheung L.K., Effect of nicotine on gene expression of angiogenic and osteogenic factors in a rabbit model of bone regeneration, Journal of Oral and Maxillofacial Surgery. 2010, 68: 777 - 781.

Ma L., Sham M.H., Zheng L. and Cheung L.K., Influence of low dose nicotine on bone healing. , J Trauma . 2010, In press.

Ma L., Zheng L., Sham M.H. and Cheung L.K., Uncoupled angiogenesis and osteogenesis in nicotine compromised bone healing, J Bone Miner Res . 2010, 25: 1305-13.

Sham M.H., Common Core Teaching Development Grant, The University of Hong Kong. 2010.

Sham M.H., Cross-talk between Hox genes and hedgehog signaling in hindbrain neurogenesis, National Institute of Biological Sciences, Beijing. 2010.

Sham M.H., Cross-talk between Hox genes and hedgehog signaling in hindbrain neurogenesis, Peking Union Medical College. 2010.

Sham M.H., Member of the Academic Editorial Board, PLoS ONE. PLoS, 2009.

Sham M.H., Outstanding Research Student Supervisor Award 2008-09, The University Of Hong Kong. 2010.

Sham M.H., Review Editor, Frontiers in Autonomic Neuroscience. Frontiers Editorial Office, 2010.

Tam C.N., Cheng M.H., Tsang S.L., Yam G.H.F., Pang C.P. and Sham M.H., Best Poster Award: Unfolded Protein Response Leading to Cataratogenesis in a Microphthalmia Cataract Mouse Mutant., 14th Research Postgraduate Symposium, December 2 & 3, 2009, The University of Hong Kong. 2009.

Tam C.N., Cheng M.H., Tsang S.L., Yam G...H...F..., Pang C...P... and Sham M.H., Best Poster Award: Unfolded protein response leading to cataractogenesis in a microphthalmia cataract mouse mutant., 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium, 15 May 2010. . 2010.

Tam C.N., Cheng M.H., Tsang S.L., Yam G.H.F., Pang C.P. and Sham M.H., Unfolded Protein Response Leading to Cataratogenesis in a Microphthalmia Cataract Mouse Mutant, 14th Research Postgraduate Symposium, December 2 & 3, 2009, The University of Hong Kong. 2009.

Tam C.N., Cheng M.H., Tsang S.L., Yam G...H...F..., Pang C...P... and Sham M.H., Unfolded protein response leading to cataractogenesis in a microphthalmia cataract mouse mutant, 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium, 15 May 2010. . 2010.

Tam C.N., Cheng M.H., Tsang S.L., Yam G.H.F., Pang C.P. and Sham M.H., Unfolded protein response leading to cataractogenesis in a microphthalmia cataract mouse mutant, Mech. Dev. . 2009-09-06, 2009, 126: S128-129.

Tao K.P., Lai S.K., Chan Y.S., Sham M.H. and Chan S.Y., Tspyl2 is involved in Cellular Stress Response and Brain Function, 14^th Research Postgraduate Symposium, HKU, 2-3 December 2009.

Wang X., Wong E.Y.M., Hui C.C. and Sham M.H., Hoxb1 Cooperates with Gli to Mediate Sonic Hedgehog Signaling in Hindbrain Neurogenesis , 14th Research Postgraduate Symposium, December 2 & 3, 2009, The University of Hong Kong. 2009.

Wang X., Wong E.Y.M., Hui C.C. and Sham M.H., Hoxb1 cooperates with GL1 to mediate sonic hedgehog signaling in hindbrain neural progenitor cell fate determination, International Society for Stem Cell Research, 8th Annual Meeting, Moscone West, San Francisco, CA, USA, June 16-19, 2010.

Wang X., Wong E.Y.M., Hui C.C. and Sham M.H., Hoxb1 cooperates with Gli to activate NKx2.2 expression in the neurogenesis of mammalian hind brain rhombomere 4, 43rd Annual Meeting for the Japanese Society of Developmental Biologists, Jointly Sponsored by the Asia-Pacific Developmental Biology Network, Kyoto, Japan, 20-23 June. 2010.

Wang X., Wong E.Y.M., Hui C.C. and Sham M.H., Hoxb1 cooperates with Gli to activate Nkx2.2 expression in the neurogenesis of mammalian hindbrain rhombomere 4, 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium, CUHK, Hong Kong, 15 May,. 2010.

Wang X., Wong Y.M.E., Hui C.C. and Sham M.H., Hoxb1 cooperates with Gli to active Nkx2.2 expression in the neurogenesis of mammalian hind brain rhombomere 4., 43rd Annual Meeting for the Japanese Society of Developmental Biologists Kyoto, Japan, 20 June 2010 . 2010.

Wang Y., Lu L., Wu M.H., Sham M.H., Chan D., Cheah K.S.E. and Cheung M.C.H., In vitro generation of Osteo-chondroprogenitors, 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium, CUHK, Hong Kong, 15 May,. 2010.

Zhang M., Lui V.C.H., Tam P.K.H. and Sham M.H., Abnormal Enteric Nervous System Development in a Sox10^NGFP Mouse Mutant, 14th Research Postgraduate Symposium, December 2 & 3, 2009, The University of Hong Kong. 2009.

Zhang M., Leung C., Lui V.C.H., Tam P.K.H. and Sham M.H., Sox10 affects enteric neural crest cells migration in a Sox10NGFP/+ mutant , 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium, CUHK, Hong Kong, 15 May,. 2010.

Zhang M., Leung C., Lui V.C.H., Tam P.K.H. and Sham M.H., Sox10 is required for proliferation and migration of enteric neural crest stem cells, International Society for Stem Cell Research, 8th Annual Meeting, Moscone West, San Francisco, CA, USA, June 16-19, 2010.

Zhang M., Leung C., Lui V.C.H., Tam P.K.H. and Sham M.H., Sox10 mediates proliferation and migration behavior of enteric neural crest cells, 43rd Annual Meeting for the Japanese Society of Developmental Biologists, Jointly Sponsored by the Asia-Pacific Developmental Biology Network, Kyoto, Japan, 20-23 June. 2010.

Researcher : Shea GKH

List of Research Outputs

Shea G.K.H., Tsui Y.P., Chan Y.S. and Shum D.K.Y., Bone marrow-derived Schwann cells achieve fate commitment – a prerequisite for remyelination therapy, Experimental Neurology. 2010, 224: 448-458.

Shea G.K.H., Tsui Y.P., Shum D.K.Y. and Chan Y.S., Fate Commitment in Bone Marrow Stromal Cell-derived Schwann Cells is Dependant on Axonal Contact, 14th Research Postgraduate Symposium, December 2 & 3, 2009, The University of Hong Kong. 2009.

Shea G.K.H., Tsui Y.P., Chan Y.S. and Shum D.K.Y., Fate commitment in bone marrow stromal cells derived Schwann cells is dependant on axonal contact, Proceedings of 32nd Annual Meeting of Japan Neuroscience Society. 2009, P2-d12.

Researcher : Shi L

List of Research Outputs

Shi L., Yu B., Yang M. and Huang J., Engineering Tumor-killing Salmonella against Breast Cancer, 14th Research Postgraduate Symposium, December 2 & 3, 2009, The University of Hong Kong. 2009.

Researcher : Shum DKY

Project Title:	30th Annual Conference of Society for Neuroscience Effects of Chondroitin Sulfate Supplements on Regeneration of Bridged Sciatic Nerves
Investigator(s):	Shum DKY
Department:	Biochemistry
Source(s) of Funding:	URC/CRCG - Conference Grants for Teaching Staff
Start Date:	11/2000

Abstract:

N/A

Project Title:	31st Annual Meeting of Society for Neuroscience Expression of Chondroitin 6-sulfotransferase mRNA and the Sulfation Product in Post-crush Sciatic Nerves of Adult Rats
Investigator(s):	Shum DKY
Department:	Biochemistry
Source(s) of Funding:	URC/CRCG - Conference Grants for Teaching Staff
Start Date:	11/2001

Abstract:

N/A

Project Title:	Expression of heparanase in the injured spinal cord
Investigator(s):	Shum DKY
Department:	Biochemistry
Source(s) of Funding:	Small Project Funding
Start Date:	11/2003

Abstract:

To determine the time course and cellular source of heparanase expression in the bridged hemicord; to determine the distribution of substrate HS in relation to the heparanase-expressing cells.

Project Title:	33rd Annual Conference of the Society for Neuroscience Spatiotemporal Expression of Chondroitin 6-Sulfotransferase (C6ST) In Regeneration of Crush-injured Sciatic Nerves of Adult Rats C6ST Expression in Sciatic Nerve Explant Culture of Adult Rats
Investigator(s):	Shum DKY
Department:	Biochemistry
Source(s) of Funding:	URC/CRCG - Conference Grants for Teaching Staff
Start Date:	11/2003

Abstract:

N/A

Project Title:	Expression of chondroitin sulfotransferases in the injured spinal cord
Investigator(s):	Shum DKY
Department:	Biochemistry
Source(s) of Funding:	Small Project Funding
Start Date:	11/2004

Abstract:

To determine the expression profiles of the chondroitin sulfotransferases (STs) with time; to map the expression of chondroitin STs in reactive astrocytes, macrophages and meningeal fibroblasts that invade the leison site; to recover chondroitin sulfates of the lesion site and surrounding glial scar for comparison of sulfation patterns with those of intact tissue.

Project Title:	Proteoglycans in Signaling Heparan Sulfate/Syndecan-1 Modulates the Balance between Proteinase and Anti-Proteinase in Bronchiectasis
Investigator(s):	Shum DKY
Department:	Biochemistry
Source(s) of Funding:	URC/CRCG - Conference Grants for Teaching Staff
Start Date:	09/2005

Abstract:

N/A

Project Title:	Roles of chondroitin sulfates in axonal growth and patterning in the developing hindbrain
Investigator(s):	Shum DKY, Chan YS, Chan WY
Department:	Biochemistry
Source(s) of Funding:	General Research Fund (GRF)
Start Date:	09/2006
Completion Date:	02/2010

Abstract:

To test for change in sulfation pattern of hindrain CS with change in expression of chondroitin sulfotransferases in the hindbrain during vestibular neurogenesis and axonal projection; to test that projection of the vestibular commissure is dependent on facilitation by C6ST expressing cells in the axon-restrictive hindbrain matrix; 2a) to trace pioneering projections of vestibular commissures through the early rat hindbrain max, and to identify the chondroitin sulfotranferase-expressing cell types on route along the projection path; 2b) pertubation of matrix CS; 2bi) by in vivo treatment of the rat hindbrain with chondroitinase ABC or CS disaccharides; 2bii) when cellular expression of C6ST is deficient in C6ST_-/- mice.

Project Title:	Heparan sulfate oligosaccharide preparations - novel therapeutics in chronic inflammation
Investigator(s):	Shum DKY
Department:	Biochemistry
Source(s) of Funding:	Innovation and Technology Fund Internship Programme
Start Date:	10/2007

Abstract:

In need of a ready source of purified human syndecan-1, we have generated a stable cell line that produces recombinant human syndecan-1. As proof of concept, heparan sulfates carried by the recombinant syndecan-1 will be targeted for binding and protecting neutrophil elastase from inhibition by anit-elastases.

Project Title:	Use of a rat model of chronic obstructive pulmonary disease to address neutrophil elastase/anti-elastase imbalance in the airways
Investigator(s):	Shum DKY, Ip MSM, Chan CH
Department:	Biochemistry
Source(s) of Funding:	Seed Funding Programme for Applied Research
Start Date:	12/2008

Abstract:

Persistent cigarette smoking plays an important role in the pathogenesis of chonic obstructive pulmonary disease (COPD). Exacerbations of COPD are associated with chronic airway inflammation and progressive airflow limitation, leading to substantial morbidity and mortality1. Although neutrophil recruitment to the inflamed airways is integral to innate immune defense, persistent tissue damage due to unopposed neutrophil elastase (NE) activity in local microenvironmentsis a hallmark of COPD2. Deficiency in alpha1-antitrypsin (alpha 1-AT), a physiological antielastase, is one of the most common inherited disorders among Caucasians and therefore has been held a cause of protease/antiprotease imbalance in the airways of Caucasian patients3. This inherited disorder is not common among COPD patients of Chinese origin. Our preliminary study of local patients (Chinese) however found NE activity that persists even with abundant alpha 1-AT in inflammatory airway secretions. We found NE in a supramolecular complex with shed syndecan-1 and this limited inhibition of the enzyme by alpha 1-AT. This situation is similar to what we have reported in patients with bronchiectasis4, 5. We hypothesize therefore that in COPD, the heparan sulfate components of shed syndecans bind and restrict NE from inhibition by alpha 1-AT, leading to unopposed NE activity as a cause of proteolytic imbalance in the airways. We have generated stable syndecan-1 transfectants of ARH-77 lymphoid cells as a source to derive competing heparan sulfate oligosaccharides against those of shed syndecans. In the study, we therefore aim : (1) to assess proteolytic imbalance occurring in the bronchial environments of patients with COPD; (2) to identify heparan oligosaccharides that displace NE from syndecan-1-bound forms and provide NE for inhibition by endogenous alpha 1-AT ; (3) to test efficacy of selected heparan oligosaccharides in restoring elastase/anti-elastase balance in airway environments of a smoke-induced rat model of COPD.

Project Title:	Role of heparanase in synaptic plasticity at the vestibular nucleus
Investigator(s):	Shum DKY, Chan YS
Department:	Biochemistry
Source(s) of Funding:	General Research Fund (GRF)
Start Date:	01/2009

Abstract:

(1) To examine spatiotemporal expression of heparanase and HS proteoglycans in relation to postnatal consolidation of perineuronal nets in the VN; (2) To determine how neuronal activity regulates heparanase release for adjustments of HS-dependent synaptic strength of VN neurons.

List of Research Outputs

Cai S., Shum D.K.Y., Chan Y.S. and Fu X.B., Outstanding Abstract Prize (Poster Category), Research Center of Heart, Brain, Hormone & Healthy Aging. 2010.

Cham W.C., Ma C.W., Zhang Y., Xie H., Yung W.H., Chan Y.S. and Shum D.K.Y., The Regulatory Role of Heparanase in Synaptic Plasiticity at Hippocampus, 14th Research Postgraduate Symposium, December 2 & 3, 2009, The University of Hong Kong. 2009.

Cham W.C., Ma C.W., Zhang Y., Xie H., Yung W.H., Chan Y.S. and Shum D.K.Y., The regulatory role of heparanase of synaptic plasticity in hippocampus, Society Neuroscience Abstract (USA). 2009, 318.3/C4.

Chan C.H., Leung V.O.Y., Lam C.L.D., Mak J.C.W., Freeman C., Ip M.S.M. and Shum D.K.Y., Sulfated maltoheptaose reduces neutrophilic airway inflammation in a smoking rat model of chronic obstructive pulmonary disease , Fifth International Symposium on Healthy Aging: “Is Aging a Disease?” 6-7 March 2010 . 2010.

Ip C.H., Chan C.H. and Shum D.K.Y., Serglycin in the HL-60 degranulation product retains its sulfated chondroitin/dermatan moieties and affinity for neutrophil elastase, 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium, CUHK, Hong Kong, 15 May,. 2010.

Lai C.H., Yiu N.S.C., Lai S.K., Ng K.P., Yung K.K., Shum D.K.Y. and Chan Y.S., Maturation of canal-related brainstem neurons in the detection of horizontal angular acceleration in rats, Journal of Comparative Neurology. 2010, 518: 1742-1763.

Li M., Chan Y.S. and Shum D.K.Y., Chondroitin sulfate moieties expressed by migrating motor neurons in explant cultures of the rat hindbrain, Proceedings of 32nd Annual Meeting of Japan Neuroscience Society . 2009, P1-e10.

Li M., Chan Y.S. and Shum D.K.Y., Expression of chondroitin sulfotransferases in cranial motor neurons in the embryonic rat hindbrain, Neuroscience Bulletin (Suppl 1). [8^th Biennial Conference of Chinese Society for Neuroscience. Guangzhou. Nov. 7-10, 2009]. 2009, 25: 113.

Ma C.W., Lai C.H., Lai S.K., Tse Y.C., Yung K.K., Shum D.K.Y. and Chan Y.S., Developmental distribution of vestibular nuclear neurons responsive to different speeds of horizontal translation, Brain Research. 2010, 1326: 62-67.

Ma C.W., Lai C.H., Chan Y.S. and Shum D.K.Y., In vitro and in vivo studies of perineuronal glycosaminoglycans in synaptic functions, Society Neuroscience Abstract (USA) . 2009, 719.10/D6.

Ma C.W., Zhang Y., Cham W.C., Chan Y.S. and Shum D.K.Y., Role of heparanase in synaptic plasticity at the hippocampus and vestibular nucleus , J. Physiological Sciences (Suppl 1). [36^th Int’l Congress of International Union of Physiological Sciences Kyoto, Japan. July 27 - Aug. 1, 2009]. 2009, 59: 145.

Ng K.P., Han L., Lau P.Y.P., Li C., Lai C.H., Shum D.K.Y. and Chan Y.S., Postnatal refinement of gravity-related spatial coding to the central vestibular circuitry, J. Physiological Sciences (Suppl 1). [36^th Int’l Congress of International Union of Physiological Sciences. Kyoto, Japan. July 27 - Aug. 1, 2009]. 2009, 59: 204.

Shea G.K.H., Tsui Y.P., Chan Y.S. and Shum D.K.Y., Bone marrow-derived Schwann cells achieve fate commitment – a prerequisite for remyelination therapy, Experimental Neurology. 2010, 224: 448-458.

Shea G.K.H., Tsui Y.P., Shum D.K.Y. and Chan Y.S., Fate Commitment in Bone Marrow Stromal Cell-derived Schwann Cells is Dependant on Axonal Contact, 14th Research Postgraduate Symposium, December 2 & 3, 2009, The University of Hong Kong. 2009.

Shea G.K.H., Tsui Y.P., Chan Y.S. and Shum D.K.Y., Fate commitment in bone marrow stromal cells derived Schwann cells is dependant on axonal contact, Proceedings of 32nd Annual Meeting of Japan Neuroscience Society. 2009, P2-d12.

Shum D.K.Y., Ip M.S.M., Chan C.H. and Leung O.Y.V., Compositions and Methods for Treating chronic Respiratory Inflammation. U.S. Provisional Application No. 61/308,597 filed on Feb 26, 2010 with the U.WS. Patent and Trademark Office. , 2010.

Tsui Y.P., Chan Y.S. and Shum D.K.Y., Bone marrow stromal cell-derived oligodendrocyte precursor cells: A new perspective in CNS myelin repair, 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium, CUHK, Hong Kong, 15 May,. 2010.

Yuen J.W.M., Gohel M...D...I..., Poon M.W., Shum D.K.Y., Tam P.C. and Au D.W.T., The initial and subsequent inflammatory events during calcium oxalate lithiasis, Clinica Chimica Acta. 2010, 411: 1018-1026.

Researcher : Shum KT

List of Research Outputs

Lin Y., Shum K.T., Tanner J.A. and Zheng B., Study of DNA aptamers binding H5N1 virus nucleoprotein. , Thailand Conference on Emerging Infectious and Neglected Diseases. Chonbrti, Thailand.. 2010.

Lui E.L.H., Shum K.T., Sam L., Wang Y., Watt R.M. and Tanner J.A., Aptamers Against Polyphosphate Kinase 2 (PPK2) From Mycobacterium Tuberculosis As A Potential Approach Of Novel Anti-Tuberculosis Therapy , CUHK Croucher Advanced Study Institute “Structure-Based Screening and Design of Ligands for Protein Targets” . 2009.

Lui L.H.E., Shum K.T. and Tanner J.A., Effect of Inorganic Polyphosphate on Higher Eukaryotic Cells, 14th Research Postgraduate Symposium, December 2 & 3, 2009, The University of Hong Kong. 2009.

Shum K.T., Chan C., Leung C.M. and Tanner J.A., Aptamer-based Inhibitor of Sclerostin for Osteoporosis Therapy, 14th Research Postgraduate Symposium, December 2 & 3, 2009, The University of Hong Kong. 2009.

Shum K.T., Lui E.L.H., Wang Y., Watt R.M. and Tanner J.A., Aptamer-mediated inhibition of Mycobacterium tuberculosis polyphosphate kinase 2. , 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium . 2010.

Shum K.T., Chan C.S.L., Leung C.M. and Tanner J.A., Aptamer-mediated inhibition of sclerostin for osteoporosis therapy. , 5th Annual Meeting of the Oligonucleotide Therapeutics Society, Fukuoka, Japan . 2009.

Shum K.T., Chan C.S.L., Leung C.M. and Tanner J.A., G-Quadruplex Aptamers Inhibit Sclerostin for Osteoporosis Therapy, CUHK Croucher Advanced Study Institute “Structure-Based Screening and Design of Ligands for Protein Targets” . 2009.

Shum K.T., Merit Award and Travel Grant for Novartis International Biotechnology Leadership Camp (BioCamp 2009), United States , Novartis Pharmaceuticals. 2009.

Shum K.T., Oligonucleotide Therapeutics Society (OTS) 2009 Travel Award , 2009.

Tanner J.A. and Shum K.T., Chemical Biology, Kirk-Othmer Encyclopedia of Chemical Technology. New York, USA, John Wiley and Sons, 2010, 27: 1-26.

Tanner J.A., Shum K.T. and Chan C.S.L., High-affinity nucleic acid aptamers against sclerostin protein, US Provisional Patent. USA, 2010, 61/349,058: 61/349,058.

Tanner J.A., Zheng B., Lin Y., Kimura M., Lui E.L.H. and Shum K.T., Selection, validation and delivery of DNA aptamers against infectious disease targets. , 5th Annual Meeting of the Oligonucleotide Therapeutics Society, Fukuoka, Japan . 2009.

Researcher : Shum KT

List of Research Outputs

Shum K.T., Merit Award and Travel Grant for Novartis International Biotechnology Leadership Camp (BioCamp 2009), United States , Novartis Pharmaceuticals. 2009.

Shum K.T., Oligonucleotide Therapeutics Society (OTS) 2009 Travel Award , 2009.

Tanner J.A. and Shum K.T., Chemical Biology, Kirk-Othmer Encyclopedia of Chemical Technology. New York, USA, John Wiley and Sons, 2010, 27: 1-26.

Tanner J.A., Shum K.T. and Chan C.S.L., High-affinity nucleic acid aptamers against sclerostin protein, US Provisional Patent. USA, 2010, 61/349,058: 61/349,058.

Researcher : Siu KL

List of Research Outputs

Ng M.H., Siu K.L., Kok K.H., Ching Y.P. and Jin D., MIP-T3 is a novel inhibitor in TBK1/IRF3-dependent antiviral response. , 28th Annual meeting of the American Society for Virology. 2009.

Ng M.H., Siu K.L., Ching Y.P. and Jin D., MIP-T3 is a novel inhibitor in TBK1/IRF3-dependent antiviral response, The American Society for Virology 28th Annual Meeting, University of British Columbia, Vancouver, BC, Canada. 2009.

Siu K.L., Chan C.P., Chan C.S., Zheng B. and Jin D., Severe acute respiratory syndrome coronavirus nucleocapsid protein does not modulate transcription of human FGL2 gene., J. Gen. Virol.. 2009, 90: 2107-2113.

Tang V.H.M., Siu K.L., Wong C.M. and Jin D., Loss of Yeast Peroxiredoxin Tsa1p Induces Genome Instability through Activation of the DNA Damage Checkpoint and Elevation of dNTP Levels, In: Orna Cohen-Fix, PLoS Genetics. 2009, 5(10): e1000697.

Researcher : Smith DK

Project Title:	Patterns in proteins with unusual flexibility profiles
Investigator(s):	Smith DK
Department:	Biochemistry
Source(s) of Funding:	Seed Funding for New Staff
Start Date:	08/2002

Abstract:

To identify what makes some proteins imcompatible with flexibility parameters calculated from a large set of protein structures.

List of Research Outputs

Jia Y., Song Y., Yiu S.M. and Smith D.K., Bioinformatics Study of the Lineage and Tissue Specificity of Gene Expression, 14th Research Postgraduate Symposium, December 2 & 3, 2009, The University of Hong Kong. 2009.

Song Y., Tang L.F., Cheung C.L., Sham P.C., McClurg P., Smith D.K., Tanner J.A., Su A.I., Cheah K.S.E. and Kung A.W.C., Genome-wide haplotype association mapping in mice identifies a genetic variant in CER1 associated with bone mineral density and fracture in southern Chinese women, The American Society of Human Genetics 59th Annual Meeting, Honolulu, Hawaii. 2009.

Researcher : Song Y

Project Title:	A Randomized Controlled Trial of the Therapeutic Effects of Tai Chi and Mahjong on Older Persons With Cognitive Impairment
Investigator(s):	Song Y
Department:	Biochemistry
Source(s) of Funding:	General Research Fund (GRF)
Start Date:	01/2008

Abstract:

1) To see if a 3-month intervention of Tai Chi only, mahjong only, and Tai Chi cum mahjong improve the cognitive, physical, behavioral, and emotional functioning of persons with cognitive impairment, and which of these interventions is most effective in which domain. 2) To see if the effects of these therapies alter the long-term trajectories of these persons into 12 months post-treatment. 3) To investigate if the beneficial effects of Tai Chi and mahjong, if any, are limited to those without the apolipoprotein E ε4 allele, a genetic marker of Alzheimer disease.

Project Title:	Fine mapping candidate loci for nasopharyngeal carcinoma (NPC) in southern Chinese specifically linked to EBV aetiopathogenesis
Investigator(s):	Song Y, Guan XY, Chua DTT
Department:	Biochemistry
Source(s) of Funding:	Research Fund for the Control of Infectious Diseases - Full Grants
Start Date:	01/2009

Abstract:

To further study these candidate loci in our dataset in order to fine mapping the candidate regions. Detecting NPC gene(s) will help diagnosis and treatment of NPC, and understand the molecular mechanisms

Project Title:	Detecting genes and functional analysis of a novel gene (Cer1) significantly associated with bone mineral density (BMD) in mice.
Investigator(s):	Song Y, Chan D, Sham PC, Tanner JA
Department:	Biochemistry
Source(s) of Funding:	General Research Fund (GRF)
Start Date:	01/2009

Abstract:

(1) Functional characterization of Cer1 in vitro to verify its positive identification of a QTL for BMD regulation; (2) Fine mapping and detecting other genes involved in regulation of BMD in mice.

Project Title:	Whole Genome-wide Association Study of Adolescent Idiopathic Scoliosis in Southern Chinese
Investigator(s):	Song Y, Cheung KMC, Sham PC, Chan D, Luk KDK
Department:	Biochemistry
Source(s) of Funding:	Seed Funding Programme for Basic Research
Start Date:	05/2009

Abstract:

The aim of this study is to systematically identify new genes associated with AIS in the southern Chinese population including previously reported linkage regions.

Project Title:	59th Annual Meeting of The American Society of Human Genetics Genome-wide haplotype association mapping in mice identifies a genetic variant in CER1 associated with bone mineral density and fracture in southern Chinese women
Investigator(s):	Song Y
Department:	Biochemistry
Source(s) of Funding:	URC/CRCG - Conference Grants for Teaching Staff
Start Date:	10/2009
Completion Date:	10/2009

Abstract:

N/A

Project Title:	The Role of Pax6 in Beta-Amyloid-induced Neuronal Apoptosis in Alzheimer's Disease
Investigator(s):	Song Y, Chu LW
Department:	Biochemistry
Source(s) of Funding:	Seed Funding Programme for Basic Research
Start Date:	01/2010

Abstract:

Alzheimer’s disease (AD) is a progressive disorder which is characterized by the neuronal dysfunction and loss in the patient brain. However, the mechanism by which neurons die in Alzheimer’s disease is not well understood. Previous reports have shown that inappropriate activation of cell cycle pathway and subsequent activation of CDK4/RB/E2F target genes contribute significantly to beta-amyloid induced neuronal death. Our previous data showed that such a downstream target gene Pax-6 might be a target gene of E2F1/c-Myb transcriptional factors in this death model. Our hypothesis is that Pax-6 may act as the downstream regulator of cell cycle pathway. Other reports also show Pax-6 contributes to involvement in apoptotic process in several death paradigms in vitro. We will examine the mechanisms by which Pax-6 regulates survival of neuronal cells upon neurotoxicity in Alzheimer’s disease model. This study may provide potential therapeutic agents and approaches for Alzheimer’s disease. Pax6 Pax-6 is a member of the paired box gene family and is expressed in the early development of the eye,pituitary, olfactory epithelium and nervous system (Stoykova A 1994). It is one of the key factors for central nervous system patterning and plays an essential role in the differentiation of cortical radial glia during development (Goetz M 1998; Simpson TI and Price DJ 2002). Pax-6 gene encodes a 422 amino acid protein consisting of two highly conserved DNA-binding domains, a bipartite paired domain (PRD) and a homeodomain (HD) and a C-terminal proline-, serine-,and threonine-rich region that functions in transcriptional activation (Walther C 1991; Epstein JA 1994). Homozygous loss of Pax6 function affects all expressing tissues and is neonatally lethal, null mutations cause aniridia in humans and Small eye (Sey) phenotype in Mice. Several upstream and intragenic Pax6 control elements have been identified, generally through transgenesis (Morgan R 2004; Takahashi JS 1994). Preliminary Studies Research in our lab focuses on the molecular mechanisms underlying Alzheimer's disease. For the previous work in our lab, to address the function of Pax6 in cell model of Alzheimer’s disease, we firstly did primary culture of cortical neurons derived from 14.5 embryonic days C57BL/6 mice, treated with exogenous Amyloid beta(A-beta) in time-dependent manner, to test whether Pax6 expression changed upon A-beta neurotoxicity. We performed Reverse Transcript PCR (RT-PCR) and Western Blot, found that Pax6 was elevated at both protein and RNA level upon A-beta treatment (Figure 1). The Pax6 expression also increases in APP overexpressing HEK293 cell lines (Swedish mutant APP (HEK293/sw))(Figure 2). We also performed survival assay of neurons, found that siRNA-mediated knockdown of Pax6 can significantly protect cultured cortical neurons from amyloid-beta induced neuronal death (Figure 3). Our previous data showed for the first time Pax6 is upregulated in neuronal death model evoked by beta-amyloid, suggested that Pax6 might be involving in the neuronal apoptosis in Alzheimer’s disease.

List of Research Outputs

Chai L., Song Y., Zee K.Y. and Leung W.K., Single nucleotide polymorphisms of complement component 5 and periodontitis, J Periodontal Res. 2010, 45(3): 301-308.

Garcia-Barcelo M.M., Yeung M.Y., Miao X.P., Tang S.M., Chen G., So M.T., Ngan E.S.W., Lui V.C.H., Chen Y., Liu X., Hui K.J.W.S., Li L., Guo W.H., Sun X.B., Tou J.F., Chan K.W., Wu X.Z., Song Y., Chan D., Cheung K.M.C., Chung P.H.Y., Wong K.K.Y., Sham P.C., Cherny S.S. and Tam P.K.H., Genome-wide association study identifies a susceptibility locus for biliary atresia on 10q24.2, Human Molecular Genetics. 2010, 19 (14): 2917-2925.

Jia Y., Song Y., Yiu S.M. and Smith D.K., Bioinformatics Study of the Lineage and Tissue Specificity of Gene Expression, 14th Research Postgraduate Symposium, December 2 & 3, 2009, The University of Hong Kong. 2009.

Kao Y.P.P., Chan D., Cheah K.S.E., Cheung K.M.C., Ho D.W.H., Karppinen J., Leong J.C.Y., Yip S.P., Song Y. and Sham P.C., Genome-wide Association Study of Degenerative Disc Disease (DDD), 14th Research Postgraduate Symposium, December 2 & 3, 2009, The University of Hong Kong. 2009.

Lam H.C., Jin D. and Song Y., Ptpn21, a Positive Mediator which Promotes ErbB4 and Suppresses Cell survival, 14th Research Postgraduate Symposium, December 2 & 3, 2009, The University of Hong Kong. 2009.

Lee K.S., Lam T.K., Song Y., Cheah K.S.E., Cheung K.M.C. and Chan D., In vivo study of Asporin, a genetic risk factor for Osteoarthritis and degenerative disc disease, 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium, CUHK, Hong Kong, 15 May,. 2010.

Song Y., Genetics of Alzheimer's Disease , Alzheimer’s Disease Conference: From Public Health to Therapeutic Insights. 2010.

Song Y., Genome-wide Association study of Scoliosis, The COTREL SCIENTIFIC ANNUAL MEETING, Paris. 2009.

Song Y., Genome-wide association study of Scoliosis in Southern Chinese. Annual Meeting of the Fondation Cotrel - Institut de France , 2009.

Song Y., Tang L.F., Cheung C.L., Sham P.C., McClurg P., Smith D.K., Tanner J.A., Su A.I., Cheah K.S.E. and Kung A.W.C., Genome-wide haplotype association mapping in mice identifies a genetic variant in CER1 associated with bone mineral density and fracture in southern Chinese women, The American Society of Human Genetics 59th Annual Meeting, Honolulu, Hawaii. 2009.

Song Y., Remmbering, Ethics in Medicine. Medical Ethics Unit, HKU, 2010, Spring 2010.

Zhang Y., Zhang Y. and Song Y., The role of Pax6 in Beta-Amyloid-induced neuronal apoptosis, 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium, CUHK, Hong Kong, 15 May,. 2010.

Researcher : Sze KL

List of Research Outputs

Sze K.L., Nagy A.N.D.R.A.S., Fosang A.M.A.N.D.A., McCulloch D.A.N.I.E.L., Cheung K.M.C. and Chan D., Poster Presentation: Impact Of Activating Adamts-5 In Cartilage-related Degenerative Disorders , UGC AoE Site Visit . 2010.

Researcher : Szeto YY

List of Research Outputs

Cheah K.S.E., Au Y.K., Szeto Y.Y., Wynn S., Chan Y.S., Cheung K.M.C., Chan W.Y., Lovell-Badge R.H., Chan D. and Fritzsch B., Molecular and developmental insights into the pathogenesis of the SOX9Y440Xcampomelic dysplasia mutation, 16th International Society of Developmental Biologists Congress 6-10th September 2009 Edinburgh International Conference Centre, UK . 2009.

Chu K.H., Zhang M., Wong E.Y.M., Szeto Y.Y., Chan Y.S., Cheah K.S.E. and Sham M.H., Best Poster Award: Sox10 mutation affects cochleo-vestibular ganglion gliogenesis, 43rd Annual Meeting for the Japanese Society of Developmental Biologists, Kyoto, Japan, 20-23 June, 2010.. 2010.

Chu K.H., Zhang M., Wong E.Y.M., Szeto Y.Y., Chan Y.S., Cheah K.S.E. and Sham M.H., Best Poster Award: Sox10 mutation affects gliogenesis of the cochleo-vestibular ganglion, 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium, 15 May 2010. 2010.

Chu K.H., Zhang M., Wong E.Y.M., Szeto Y.Y., Chan Y.S., Cheah K.S.E. and Sham M.H., Sox10 mutation affects cochleo-vestibular ganglion gliogenesis, 43rd Annual Meeting for the Japanese Society of Developmental Biologists, Kyoto, Japan, 20-23 June. 2010.

Chu K.H., Zhang M., Wong E.Y.M., Szeto Y.Y., Chan Y.S., Cheah K.S.E. and Sham M.H., Sox10 mutation affects gliogenesis of the cochleo-vestibular ganglion, 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium, 15 May 2010.

Mak A.C.Y., Szeto Y.Y., Fritzsch B. and Cheah K.S.E., Differential and overlapping expression pattern of SOX2 and SOX9 in inner ear development, Gene Expression Patterns. 2009, 9: 444-453.

Researcher : Tai CP

Project Title:	Identification of genes regulated by SOX10 that mediate cell migration
Investigator(s):	Tai CP, Cheah KSE, Cheung MCH
Department:	Biochemistry
Source(s) of Funding:	Small Project Funding
Start Date:	09/2008
Completion Date:	02/2010

Abstract:

Neural crest cells can give rise to neurons, glia, melanocytes, endocrine and mesenchymal derivatives depending on their anterior-posterior position. Basically, neural crest cells have three important roles during development. First, formation of neurons and glia in the peripheral neural system establishes links to the central nervous system. Second, neural crest cells can differentiate into melanocytes for skin protection from external conditions such as UV radiation. Finally, neural crest cells form the complicated cranial-facial structures (Le Douarin et al., 2004). The ability of neural crest cells to differentiate into diverse derivatives require the coordinated activities of different families of transcription factors involved in neural crest induction, survival and migration to the proper location for their subsequent differentiation. Among all the transcription factors characterized so far, members of group E SOX proteins, SOX9 and SOX10, have been extensively studied and shown to control many aspects of neural crest cells development. Sox9 and Sox10 are initially expressed in an overlapping manner at the premigratory stage. Following neural crest delamination, Sox9 expression is extinguished and SOX10 expression is maintained in migrating neural crest cells and later remains in specific lineages subpopulations where it has an essential role in the differentiation process. SOX10 is the first SOX protein demonstrated to be associated with neural crest development. The Sox10 dom mouse mutant and human patients with SOX10 mutations showed the phenotypes of Hirschsprung disease and Waardenburg syndromes, with absence of enteric ganglia in a variable portion of the distal colon and deafness and pigmentation defects ((Southard-Smith et al.,1998; Pingault et al.,1998; Herbarth et al., 1998)). This indicates that SOX10 affects a wide range of the neural crest-derived lineages including neuronal/glial and melanocyte. Further studies in other vertebrates suggested that SOX10 may also be involved in the specification, survival, delamination, multipotency and migration of neural crest cells (Cheung and Briscoe,2003; McKeown et al., 2005; Dutton et al., 2001). Similarly, substantial evidence from studies of several different vertebrates also support Sox9 as an important regulator in neural crest development apart from its famous roles in cartilage differentiation and sex determination. Mutations of Sox9/SOX9 in mouse and human causes Campomelic Dysplasia characterized with cleft palate, skeleton malformation and sometimes sex reversal (Wagner et al., 1994; Foster et al., 1994). Until recently, its roles in neural crest development have been demonstrated using gain-and loss-of function studies in several vertebrate species including zebrafish, Xenopus, chick and mouse (Spokony et al., 2002; McKeown et al., 2005; Cheung and Briscoe,2003; Cheung et al., 2005) Forced expression of SOX9 or Sox10 in the chick neural tube induces the expression of neural crest markers in cells that would normally generate CNS neurons. In mice deficient for Sox9, trunk neural crest cells are still generated but these cells undergo apoptosis shortly before or after emigration, suggesting that Sox9 is required for neural crest survival. It is possible that Sox10, which has overlapping expression with Sox9, compensates for its loss, thus masking an early role in mouse neural crest specification. However, increasing evidence indicate that the extent of their functional equivalence appears to be partial. Functional differences between Sox9 and Sox10 become obvious at later time point upon overexpression. Forced expression of SOX10 at 36 hours post-electroporation in the chick neural tube induces ectopic cell delamination throughout the dorsal/ventral axis, whereas the migratory behavior of Sox9 transfected cells were less dramatic Consistently, forced expression of SOX10, but not Sox9 , in lateral plate mesoderm and third branchial arch induced abnormal cell migration in mouse (Tai, 2006) These gain-of-function studies strongly suggest a potential role of SOX10 in neural crest migration and probably in other abnormal cell migration processes such as metastasis. However, the molecular mechanisms of how SOX10 can induce cell migration are largely unknown. Cell migration is a complicated process which involves cell polarization, protrusion, traction and retraction – a cycle of cell migration. Each step in this cycle involves enormous changes inside the cells such as dynamics of the cytoskeleton, expression of receptors and internalization of the receptors. The distinct expression of SOX10 in migrating neural crest cells and its specific influence on cells migration upon overexpression allow us to hypothesize that SOX10 regulates distinct genes from SOX9 and these genes confer migratory ability – coordinating cells entering the migration cycle. In order to test these hypotheses we aim to:, 1) determine the changes of target genes associated with over-expression of SOX9 and SOX10 2) identify potential target genes that are commonly or differentially regulated by SOX9 and SOX10 3) identify target genes of SOX10 that mediate cell migration References: Foster, J.W., Dominguez-Steglich, M.A., Guioli, S., Kowk, G., Weller, P.A., Stevanović, M., Weissenbach, J., Mansour, S., Young, I.D., Goodfellow, P.N., et al. (1994) Campomelic dysplasia and autosomal sex reversal caused by mutations in an SRY-related gene. Nature 372, 525-530 Wagner,T.,Wirth,J.,Meyer,J.,Zabel,B.,Held,M.,Zimmer,J.,Pasantes.J.,Bricarelli,F.D., Keutel,J.,Hustert,E.et al (1994) Autosomal sex reversal and campomelic dysplasia are caused by mutations in and around the SRY-related gene SOX9. Cell 79,1111-1120 Cheung M, Briscoe J.(2003) Neural crest development is regulated by the transcription factor Sox9. Development. 130, 5681-5693. Cheung M, Chaboissier MC, Mynett A, Hirst E, Schedl A, Briscoe J. (2005) The transcriptional control of trunk neural crest induction, survival, and delamination. Dev. Cell 8, 179-192 Dutton KA, Pauliny A, Lopes SS, Elworthy S, Carney TJ, Rauch J, Geisler R, Haffter P, Kelsh RN.(2001) Zebrafish colourless encodes sox10 and specifies non-ectomesenchymal neural crest fates. Development 128, 4113-4125 Le Douarin NM, Creuzet S, Couly G, Dupin E.(2004) Neural crest cell plasticity and its limits. Development 131, 4637-4650 Southard-Smith EM, Kos L, Pavan WJ. (1998) Sox10 mutation disrupts neural crest development in Dom Hirschsprung mouse model. Nat. Genet. 18, 60-64 Pingault V, Bondurand N, Kuhlbrodt K, Goerich DE, Préhu MO, Puliti A, Herbarth B, Hermans-Borgmeyer I, Legius E, Matthijs G, Amiel J, Lyonnet S, Ceccherini I, Romeo G, Smith JC, Read AP, Wegner M, Goossens M. (1998) SOX10 mutations in patients with Waardenburg-Hirschsprung disease. Nat. Genet. 18, 171-173. Herbarth B, Pingault V, Bondurand N, Kuhlbrodt K, Hermans-Borgmeyer I, Puliti A, Lemort N, Goossens M, Wegner M.(1998) Mutation of the Sry-related Sox10 gene in Dominant megacolon, a mouse model for human Hirschsprung disease. Proc. Natl. Acad. Sci. U.S.A.95, 5161-5165. McKeown SJ, Lee VM, Bronner-Fraser M, Newgreen DF, Farlie PG.(2005) Sox10 overexpression induces neural crest-like cells from all dorsoventral levels of the neural tube but inhibits differentiation. Dev Dyn. 233,430-444 Spokony RF, Aoki Y, Saint-Germain N, Magner-Fink E, Saint-Jeannet JP. (2002) The transcription factor Sox9 is required for cranial neural crest development in Xenopus.Development. 129, 421-432 Tai C.P. (2006) An in vivo analysis of specificity of gene transactivation by SOX proteins The university of Hong Kong

Researcher : Tam CN

List of Research Outputs

Tam C.N., Cheng M.H., Tsang S.L., Yam G.H.F., Pang C.P. and Sham M.H., Best Poster Award: Unfolded Protein Response Leading to Cataratogenesis in a Microphthalmia Cataract Mouse Mutant., 14th Research Postgraduate Symposium, December 2 & 3, 2009, The University of Hong Kong. 2009.

Tam C.N., Cheng M.H., Tsang S.L., Yam G...H...F..., Pang C...P... and Sham M.H., Best Poster Award: Unfolded protein response leading to cataractogenesis in a microphthalmia cataract mouse mutant., 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium, 15 May 2010. . 2010.

Tam C.N., Cheng M.H., Tsang S.L., Yam G.H.F., Pang C.P. and Sham M.H., Unfolded Protein Response Leading to Cataratogenesis in a Microphthalmia Cataract Mouse Mutant, 14th Research Postgraduate Symposium, December 2 & 3, 2009, The University of Hong Kong. 2009.

Tam C.N., Cheng M.H., Tsang S.L., Yam G...H...F..., Pang C...P... and Sham M.H., Unfolded protein response leading to cataractogenesis in a microphthalmia cataract mouse mutant, 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium, 15 May 2010. . 2010.

Tam C.N., Cheng M.H., Tsang S.L., Yam G.H.F., Pang C.P. and Sham M.H., Unfolded protein response leading to cataractogenesis in a microphthalmia cataract mouse mutant, Mech. Dev. . 2009-09-06, 2009, 126: S128-129.

Researcher : Tam CN

List of Research Outputs

Researcher : Tam JWO

Project Title:	14th Annual San Diego Conference: Nucleic Acid Technologies in Disease Detection Direct Flow-Through Hybridisation Device: An Effective and Lost Cost Apparatus for Rapid DNA Analysis
Investigator(s):	Tam JWO
Department:	Biochemistry
Source(s) of Funding:	URC/CRCG - Conference Grants for Teaching Staff
Start Date:	11/1999

Abstract:

N/A

Researcher : Tam PPL

List of Research Outputs

Gao Y., Leung W.L., Wong S.Y.Y., Tam P.P.L., Chan D. and Cheah K.S.E., Type IIA Procollagen: A Positive Regulator of Nodal Signaling During Early Vertebrate Embryogenesis, 14th Research Postgraduate Symposium, December 2 & 3, 2009, The University of Hong Kong. 2009.

Gao Y., Leung W.L., Wong Y.Y., Chan D., Tam P.P.L. and Cheah K.S.E., Type IIA procollagen: A positive regulator of nodal signaling during embryogenesis, 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium, CUHK, Hong Kong, 15 May. 2010.

Researcher : Tan Z

List of Research Outputs

Cheah K.S.E., Wang C., Tan Z., Melhado I.G., Niu B., Tsang K.Y., Zhang M...Q..., Ron D... and Chan D., Alleviating ER stress in chondrocytes: survival strategies with double-edged developmental consequences , 43rd Annual Meeting for the Japanese Society of Developmental Biologists Jointly Sponsored by the Asia-Pacific Developmental Biology Network, Kyoto, Japan, 20 June 2010.

Cheah K.S.E., Wang C., Tan Z., Melhado I.G., Niu B., Tsang K.Y., Zhang M...Q..., Ron D. and Chan D., Genetic and genomic analyses of chondrocyte adaptation to ER stress, The Genetics Society Spring Meeting “Mouse Genetics – Think Globally, Act Locally” The Wellcome Trust Conference Centre, Hinxton, UK, 23 April 2010 . 2010.

Tan Z., Niu B., Tsang K.Y., Melhado I.G., Zhang M., Chan D. and Cheah K.S.E., Global gene expression changes during chondrocyte adaptation to ER stress, 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium, CUHK, Hong Kong, 15 May,. 2010.

Tan Z., Niu B., Chan D. and Cheah K.S.E., Mechanisms Underlying Chondrocyte Reprogramming Surviving ER Stress, 14th Research Postgraduate Symposium, December 2 & 3, 2009, The University of Hong Kong. 2009.

Researcher : Tang HC

List of Research Outputs

Tang H.C., Yang Y., Chan D. and Cheah K.S.E., Developmental Origins of Osteoblasts in Endochondral Bone Formation, 14th Research Postgraduate Symposium, December 2 & 3, 2009, The University of Hong Kong. 2009.

Tang H.C., Yang L., Tsang K.Y., Chan D. and Cheah K.S.E., Dual origin of osteoblasts in bone formation - terminally differentiated chondrocytes contribute to bone formation in vivo, 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium, CUHK, Hong Kong, 15 May,. 2010.

Researcher : Tang HMV

List of Research Outputs

Tang H.M.V., Characterization of Yeast Peroxiredoxin tsaip In DNA Damage Response, PhD Thesis. 2009.

Researcher : Tang LF

List of Research Outputs

Song Y., Tang L.F., Cheung C.L., Sham P.C., McClurg P., Smith D.K., Tanner J.A., Su A.I., Cheah K.S.E. and Kung A.W.C., Genome-wide haplotype association mapping in mice identifies a genetic variant in CER1 associated with bone mineral density and fracture in southern Chinese women, The American Society of Human Genetics 59th Annual Meeting, Honolulu, Hawaii. 2009.

Tang L.F., Dissecting The Genetics of Complex Trait in Mouse - An Attempt Using Public resources and In-house Knockout, PhD Thesis. 2010.

Researcher : Tang VHM

List of Research Outputs

Tang V.H.M., Siu K.L., Wong C.M. and Jin D., Loss of Yeast Peroxiredoxin Tsa1p Induces Genome Instability through Activation of the DNA Damage Checkpoint and Elevation of dNTP Levels, In: Orna Cohen-Fix, PLoS Genetics. 2009, 5(10): e1000697.

Tang V.H.M., Kong K.Y., Wong G.W., Qiu H.F., Jin D. and Hinnebusch A.G., Yeast arginine methyltransferase Hmt1p is required for transcription elongation and termination, Mechanisms of Eukaryotic Transcription, Cold Spring Harbor Laboratory. 2009.

Wong C.M., Tang V.H.M., Kong K.Y., Wong G.W., Qiu H., Jin D. and Hinnebusch A.G., Yeast arginine methyltransferase Hmt1p regulates transcription elongation and termination by methylating Npl3p, Nucleic Acids Research. 2010, 38(7): 2217-2228.

Researcher : Tanner JA

Project Title:	Comparative Characterization of the Two Polyphosphate Kinases of M. tuberculosis
Investigator(s):	Tanner JA
Department:	Biochemistry
Source(s) of Funding:	Seed Funding Programme for Basic Research
Start Date:	02/2005

Abstract:

Inorganic polyphosphate (polyP) is present in every cell in nature and consists of chains of tens or hundreds of orthophosphate residues linked by high-energy phospoanhydride bonds. For decades, the molecule was ignored as a 'molecular fossil', but recent discoveries, principally by Nobel Prize winner Arthur Kornberg, have challenged this view and brought deserved attention to this forgotten biopolymer [1]. In prokaryotes, the molecule plays a critical role in physiological adjustments to growth, development, stress and deprivation [2], besides acting as a phosphate reservoir, a metal chelator, a buffer, and is an important factor in mRNA processing and degradadation. In eukaryotes, roles have only started to be uncovered in the last year or two, but the molecule has already been shown to be a regulatory factor in the proliferative signaling pathways of mammalian cells [3]. It is clear that this simple inorganic molecule plays fundamental and crucial roles that are only just beginning to be unravelled. Until 2002, it was believed that polyphosphates were synthesized by a single class of enzymes, the polyphosphate kinases (PPK). However, a landmark paper from Kornberg in 2002 challenged that view [4], and it was discovered that there are in fact two classes of polyphosphate synthetases in prokaryotes: PPK1 and PPK2. Some bacteria, such as E. coli and H. pylori, only possess PPK1, others, such as M. tuberculosis, have both PPK1 and PPK2, whilst a few others only have PPK2. As PPK1 and PPK2 only exist in microorganisms and are absolutely absent from higher eukaryotes, these are excellent targets for new classes of antibacterials. As a first step towards both new antibacterials against tuberculosis and to deepen our understanding of this fundamental class of enzymes, we here propose to purify and perform a comparative characterization of PPK1 and PPK2 from M. tuberculosis. We shall test the emerging hypothesis that PPK1 is primarily a Mg2+-dependent ATP driven kinase whilst PPK2 is primarily a Mn2+-dependent GTP driven kinase. If this hypothesis holds true, it carries significant insight into the relationship between inorganic polyphosphate and the cell cycle. We can break down the overall objectives of this seed project funding grant into a three-stage process: I. Cloning of the ppk1 and ppk2 genes from M. tuberculosis. II. Purification of the PPK1 and PPK2 proteins. III. Comparative enzymatic characterization of PPK1 and PPK2. 1. Kornberg, A., N. N. Rao, et al. (1999). "Inorganic polyphosphate: a molecule of many functions." Annu Rev Biochem 68: 89-125. 2. Kuroda, A., K. Nomura, et al. (2001). "Role of inorganic polyphosphate in promoting ribosomal protein degradation by the Lon protease in E. coli." Science 293(5530): 705-8. 3. Wang, L., C. D. Fraley, et al. (2003). "Inorganic polyphosphate stimulates mammalian TOR, a kinase involved in the proliferation of mammary cancer cells." Proc Natl Acad Sci U S A 100(20): 11249-54. 4. Zhang, H., K. Ishige, et al. (2002). "A polyphosphate kinase (PPK2) widely conserved in bacteria." Proc Natl Acad Sci U S A 99(26): 16678-83.

Project Title:	Mechanistic Insight into Polyphosphate Kinase 2 - a Fundamental Enzyme of Polyphosphate Catabolism
Investigator(s):	Tanner JA
Department:	Biochemistry
Source(s) of Funding:	Seed Funding Programme for Basic Research
Start Date:	04/2006

Abstract:

Long polyphosphate chains were long thought to be irrelevant to biology and to be a by-product of metabolism, but recent discoveries have overhauled this view and have refocused attention on these fundamental simple molecules [1]. Polyphosphates act in prokaryotes as a control of growth, development, stress, an energy source, a buffer, a metal chelator, and play a role in RNA processing [2]. Perhaps even more significantly, in eukaryotes the molecules have been identified as being critical to certain proliferative signalling pathways of mammalian cells [3]. Two classes of enzymes have been discovered that synthesise these molecules from ATP – polyphosphate kinase 1 (PPK1) and polyphosphate kinase 2 (PPK2) [4]. Polyphosphates are simple and fundamental molecules, yet their roles remain poorly investigated, despite the possibility that structural and functional variation as introduced by branching could result in a completely new class of informational biological macromolecule. Last year, our team was awarded a grant from the Seed Funding for Basic Research scheme entitled “Comparative Characterisation of the Two Polyphosphate Kinases of M. tuberculosis”. The aim of that grant was to clone, purify and characterise PPK1 and PPK2 from Mycobacterium tuberculosis (MTB) with a view to drug development by inhibiting these enzymes (in a follow-up RGC grant application). Our initial hypothesis was that PPK1 was an ATP-driven and Mg2+-dependent polyphosphate kinase, whilst PPK2 was a GTP-driven and Mn2+-dependent polyphosphate kinase. Our research for that grant was successful in that we were able to test the hypothesis, but we actually found that PPK2 was far more effective in catalysing the reverse reaction of hydrolysing long-chain polyphosphate in the presence of GDP. This suggests a novel mechanism for forming energy rich nucleoside triphosphates (and possible tetraphosphates), and is a fascinating departure from our initial hypothesis. However, this opens up many fascinating questions but unfortunately meant that our research was premature for submission to the RGC in the 2005 round. With a view to submitting this research in the 2006 RGC round, here, our overall objective is to strengthen our hypothesis that PPK2 provides a novel route to nucleoside triphosphates from long-chain polyphosphates. We will achieve our objective in three stages: 1. Investigate whether GTP or Gp4 is generated during PPK2 mediated polyphosphate hydrolysis. At present, we use a gel-based assay that enables us to observe changes in polyphosphate chain length. It remains unclear whether phosphate or pyrophosphate is transferred to GDP, and hence whether GTP or Gp4 is generated during the reaction. This is essential mechanistic information prior to observing the details of the kinetics of PPK2 in stage 2. 2. Measure the precise kinetics of GTP/Gp4 formation and polyphosphate catabolism by PPK2 We will use various assays to examine the efficacy of GTP/Gp4 synthesis and the rates of polyphosphate catabolism, and compare these to published kinetic data for polyphosphate formation by PPK1. 3. Measure levels of polyphosphate in a cellular system comparing cells overexpressing PPK1 and PPK2. If our hypothesis holds true that PPK1 plays an anabolic role, whilst PPK2 plays a catabolic role, then we should expect that a system overexpressing PPK1 should have high polyphosphate levels, whilst a system overexpressing PPK2 should have low polyphosphate levels. It would be difficult for technical reasons to test this actually in MTB, but E. coli will act as an excellent model system for the function of the two enzymes. We will use cells overexpressing either MTB PPK1 or PPK2, and measure cellular polyphosphate levels under a variety of conditions. Stage 2 will provide enzymatic, and stage 3 will provide cellular evidence that support our revised hypothesis for PPK2 function. Besides being scientifically important with regards to our understanding of the roles of polyphosphate within the cell, these enzymes are excellent drug targets for development of new classes of MTB drugs. This will provide a firm foundation for a later submission to the RGC in 2006 to discover inhibitors of PPK2 for drug development against MTB. 1. Kornberg, A., N. N. Rao, et al. (1999). "Inorganic polyphosphate: a molecule of many functions." Annu Rev Biochem 68: 89-125. 2. Kuroda, A., K. Nomura, et al. (2001). "Role of inorganic polyphosphate in promoting ribosomal protein degradation by the Lon protease in E. coli." Science 293(5530): 705-8. 3. Wang, L., C. D. Fraley, et al. (2003). "Inorganic polyphosphate stimulates mammalian TOR, a kinase involved in the proliferation of mammary cancer cells." Proc Natl Acad Sci U S A 100(20): 11249-54. 4. Zhang, H., K. Ishige, et al. (2002). "A polyphosphate kinase (PPK2) widely conserved in bacteria." Proc Natl Acad Sci U S A 99(26): 16678-83.

Project Title:	Identification, validation and characterization of the eukaryotic enzymes and regulators of inorganic polyphosphate metabolism
Investigator(s):	Tanner JA
Department:	Biochemistry
Source(s) of Funding:	General Research Fund (GRF)
Start Date:	01/2008

Abstract:

1. Development of polyphosphate kinase-based assays for detailed analysis of polyphosphate levels optimized for application to mammalian cell culture. 2. Three-pronged approach to determination of mammalian polyphosphate function by: a. impact of modulated polyphosphate levels on cellular differentiation b. determination of polyphosphate-controlled gene expression c. determination of polyphosphate interacting protein partners. 3. Determination of polyphosphate related protein candidate function in mammalian cell lines by cell biology studies.

Project Title:	Development of an Aptamer-Nanoparticle Based Rapid Diagnostic Test for Malaria
Investigator(s):	Tanner JA, Sun H
Department:	Biochemistry
Source(s) of Funding:	General Research Fund (GRF)
Start Date:	01/2009

Abstract:

(1) Stage I: Express and purify the malarial antigen histidine-rich protein (HRP II); (2) Stage II: Evolve DNA-based aptamers against the HRP II using in vitro evolution methods established in our laboratory: (3) Stage III: Validate and characterize aptamer binding to the histidine-rich protein 2 using a battery of biochemical and biophysical tests.

Project Title:	Targeting SATB1 with Aptamers for Breast Cancer Therapy
Investigator(s):	Tanner JA
Department:	Biochemistry
Source(s) of Funding:	Seed Funding Programme for Applied Research
Start Date:	06/2009

Abstract:

Aptamers are in vitro evolved nucleic acids that bind specifically and tightly to macromolecular targets, thereby inhibiting their function. Aptamers have already had major impact in contemporary pharmaceuticals, for example they are used clinically for the treatment of macular degeneration (Macugen). One current drawback of aptamers is that they are typically used against extracellular targets as crossing the cell membrane is a significant challenge. In our recent CERG grant to use aptamers to target the SARS coronavirus helicase, we managed to solve this challenge by delivering the aptamers across the membrane using a novel liposomal delivery system as evidenced by fluorescently labelled aptamers. However, we also observed that the aptamers migrated specifically to the nucleus. This was a problem for inhibiting the cellular function of the cytoplasmic localized SCV helicase, but presents us an opportunity to use this technology to specifically modulate the function of a nuclear protein. Inhibition of nuclear proteins by small molecules has been a significant challenge, despite the fact that often such transcription factors could be a critical approach for therapy seeing as they often sit at the top of large regulatory networks. Here, we propose to tailor our new aptamer delivery technology to inhibit the function of SATB1, a transcription factor that is a master regulator of various processes in the development of metastatic breast cancer. Special AT-rich protein 1 (SATB1) is a nuclear protein that functions as a genome organizer, and was originally characterized as a transcription factor important for proper T-cell development[1]. It binds to multiple genomic loci containing AT-rich DNA sequences, and by recruiting chromatin-remodeling enzymes it regulates chromatin structure and gene expression[2]. Very recently in 2008 a particularly important article was released in Nature, showing that SATB1 reprogrammes gene expression to promote breast tumour growth and metastasis [3]. SATB1 is expressed by aggressive breast cancer cells and acts as a master regulator to change the expression of over 1000 genes. Induced expression led to a breast cancer phenotype promoting the growth of large tumours, and conversely knockdown of this protein using short hairpin RNA in late stage metastatic breast cancer cell lines yielded inhibition of anchorage-independent growth. SATB1 controls specific epigenetic changes that occur in breast cancer and this protein is now one of the most important targets for the development of therapy against breast cancer. However, inhibiting a transcription factor with typical small molecule drug-like molecules has long been known as a particular challenging task. Aptamers are particularly suited to inhibiting a transcription factor as they are able to block nucleic acid binding sites by using their bulky surface and their similar biophysical properties. We have a delivery system for specific nuclear delivery which gives us an international competitive advantage for successfully targeting this protein. Therefore, we will bring together our technology with this critically important drug target, which will have important implications not only for breast cancer, but has long-term ambitions to provide a solution for transcription factor function modulation. The applied applications to both medicine and biotechnology of such work, if successful, are very significant. The extent of this particular seed funding proposal is certainly achievable: we intend to purify the SATB1 protein, develop aptamers against the protein, then characterise the biophysical interactions between the two. This will set the ball rolling for a full application to the ITF for more significant funding to investigate their actual efficacy in breast cancer cell lines and cell models. We can structure our objectives into three simple steps for this seed funding grant: Stage I: Express and purify target protein SATB1 Stage II: Select aptamers against SATB1 using in-house validated aptamer selection approaches Stage III: Validate and characterize the selected aptamers using various biophysical tests to enable optimization for cell culture work in later grant Successful completion of these three stages will lay the foundation for applying for the appropriate external funding which would be required to thoroughly test this approach against breast cancer in cell culture and in animal models. * All references shown at end of chapter entitled Research Plan and Methodology

Project Title:	5th Annual Meeting of the Oligonucleotide Therapeutics Society SELECTION, VALIDATION AND DELIVERY OF DNA APTAMERS AGAINST INFECTIOUS DISEASE TARGETS
Investigator(s):	Tanner JA
Department:	Biochemistry
Source(s) of Funding:	URC/CRCG - Conference Grants for Teaching Staff
Start Date:	11/2009
Completion Date:	11/2009

Abstract:

N/A

Project Title:	New approaches in the nuclear delivery and targeting of aptamers to augment osteoblast activity for osteoporosis
Investigator(s):	Tanner JA, Chan D
Department:	Biochemistry
Source(s) of Funding:	General Research Fund (GRF)
Start Date:	01/2010

Abstract:

1) Stage I: Express and purify the WWP1 E3 ligase, the osteoporosis drug target that is a regulator of Runx2 levels; 2) Stage II: Evolve DNA-based aptamers against WWP1 using in vitro evolution methods established in our laboratory; 3) Stage III: Validate and characterize aptamer binding to WWP1 using various biochemical and biophysical tests; 4) Stage IV: Investigate stabilization and delivery of WWP1 aptamers to the nucleus and cellular impact of these aptamers in an osteoblast cell culture model.

List of Research Outputs

Cheung Y.W. and Tanner J.A., Aptamers against P. falciparum lactate dehydrogenase as a new approach to malaria diagnosis. , CUHK Croucher Advanced Study Institute “Structure-Based Screening and Design of Ligands for Protein Targets” . 2009.

Cheung Y.W. and Tanner J.A., Improving Method for Malaria Detection - An Aptamer Approach, 14th Research Postgraduate Symposium, December 2 & 3, 2009, The University of Hong Kong. 2009.

Choi M.Y., Chan C.C.Y., Chan D., Luk K.D.K., Cheah K.S.E. and Tanner J.A., Biochemical consequences of sedlin mutations that cause spondyloepiphyseal dysplasia tarda, Biochemical Journal. Great Britain, Biochemical Society, 2009, 423: 233-242.

Huang J., Sun H., Tanner J.A. and Watt R.M., Determine the functions of the putative metal-binding domain of SARS-CoV helicase, Hong Kong Medical Journal. 2009, 15(5) Supplement: 6:15-16.

Lin Y., Tanner J.A. and Zheng B., In vivo selection and characterization of DNA aptamers against H5N1 virus nucleoprotein. , European Congress of Clinical Microbiology and Infectious Diseases. Vienna, Austria. 2010.

Lin Y., Shum K.T., Tanner J.A. and Zheng B., Study of DNA aptamers binding H5N1 virus nucleoprotein. , Thailand Conference on Emerging Infectious and Neglected Diseases. Chonbrti, Thailand.. 2010.

Lui E.L.H., Shum K.T., Sam L., Wang Y., Watt R.M. and Tanner J.A., Aptamers Against Polyphosphate Kinase 2 (PPK2) From Mycobacterium Tuberculosis As A Potential Approach Of Novel Anti-Tuberculosis Therapy , CUHK Croucher Advanced Study Institute “Structure-Based Screening and Design of Ligands for Protein Targets” . 2009.

Lui E.L.H., Li L. and Tanner J.A., Impact of Extracellular Inorganic Polyphosphate on Osteoblast Proliferation, Differentiation and Apoptosis. , 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium . 2010.

Lui L.H.E., Shum K.T. and Tanner J.A., Effect of Inorganic Polyphosphate on Higher Eukaryotic Cells, 14th Research Postgraduate Symposium, December 2 & 3, 2009, The University of Hong Kong. 2009.

Shum K.T., Chan C., Leung C.M. and Tanner J.A., Aptamer-based Inhibitor of Sclerostin for Osteoporosis Therapy, 14th Research Postgraduate Symposium, December 2 & 3, 2009, The University of Hong Kong. 2009.

Shum K.T., Lui E.L.H., Wang Y., Watt R.M. and Tanner J.A., Aptamer-mediated inhibition of Mycobacterium tuberculosis polyphosphate kinase 2. , 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium . 2010.

Shum K.T., Chan C.S.L., Leung C.M. and Tanner J.A., Aptamer-mediated inhibition of sclerostin for osteoporosis therapy. , 5th Annual Meeting of the Oligonucleotide Therapeutics Society, Fukuoka, Japan . 2009.

Shum K.T., Chan C.S.L., Leung C.M. and Tanner J.A., G-Quadruplex Aptamers Inhibit Sclerostin for Osteoporosis Therapy, CUHK Croucher Advanced Study Institute “Structure-Based Screening and Design of Ligands for Protein Targets” . 2009.

Song Y., Tang L.F., Cheung C.L., Sham P.C., McClurg P., Smith D.K., Tanner J.A., Su A.I., Cheah K.S.E. and Kung A.W.C., Genome-wide haplotype association mapping in mice identifies a genetic variant in CER1 associated with bone mineral density and fracture in southern Chinese women, The American Society of Human Genetics 59th Annual Meeting, Honolulu, Hawaii. 2009.

Tanner J.A. and Shum K.T., Chemical Biology, Kirk-Othmer Encyclopedia of Chemical Technology. New York, USA, John Wiley and Sons, 2010, 27: 1-26.

Tanner J.A. and Watt R.M., Common Core Teaching Development Grant, 2010.

Tanner J.A., Shum K.T. and Chan C.S.L., High-affinity nucleic acid aptamers against sclerostin protein, US Provisional Patent. USA, 2010, 61/349,058: 61/349,058.

Tanner J.A., Zheng B., Lin Y., Kimura M., Lui E.L.H. and Shum K.T., Selection, validation and delivery of DNA aptamers against infectious disease targets. , 5th Annual Meeting of the Oligonucleotide Therapeutics Society, Fukuoka, Japan . 2009.

Tucker W. and Tanner J.A., Aptameric Inhibition of E3 Ubiquitin Ligase WWP1 for Osteoporosis Therapy. , 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium . 2010.

Researcher : Tsang KY

List of Research Outputs

Cheah K.S.E., Wang C., Tan Z., Melhado I.G., Niu B., Tsang K.Y., Zhang M...Q..., Ron D... and Chan D., Alleviating ER stress in chondrocytes: survival strategies with double-edged developmental consequences , 43rd Annual Meeting for the Japanese Society of Developmental Biologists Jointly Sponsored by the Asia-Pacific Developmental Biology Network, Kyoto, Japan, 20 June 2010.

Cheah K.S.E., Wang C., Tan Z., Melhado I.G., Niu B., Tsang K.Y., Zhang M...Q..., Ron D. and Chan D., Genetic and genomic analyses of chondrocyte adaptation to ER stress, The Genetics Society Spring Meeting “Mouse Genetics – Think Globally, Act Locally” The Wellcome Trust Conference Centre, Hinxton, UK, 23 April 2010 . 2010.

Lie Y.S., Chan W.C.W., Tsang K.Y., Cheah K.S.E. and Chan D., Impact of intracellular clearance of unfolded proteins on chondrocytes cell fate, 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium, CUHK, Hong Kong, 15 May,. 2010.

Tan Z., Niu B., Tsang K.Y., Melhado I.G., Zhang M., Chan D. and Cheah K.S.E., Global gene expression changes during chondrocyte adaptation to ER stress, 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium, CUHK, Hong Kong, 15 May,. 2010.

Tang H.C., Yang L., Tsang K.Y., Chan D. and Cheah K.S.E., Dual origin of osteoblasts in bone formation - terminally differentiated chondrocytes contribute to bone formation in vivo, 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium, CUHK, Hong Kong, 15 May,. 2010.

Tsang K.Y., Chan D., Bateman J.F. and Cheah K.S.E., In vivo cellular adaptation to ER stress: survival strategies with double-edged consequences, J. Cell Science. 2010, 123: 2145-21554.

Tsang K.Y., Cheung K.M.C., Chan D. and Cheah K.S.E., The developmental roles of the extracellular matrix: beyond structure to regulation, Cell Tissue Res. 2010, 339(1): 93-110.

Researcher : Tsang SL

List of Research Outputs

Chen M., Huang J., Hu L., Zheng B., Chen L., Tsang S.L. and Guan X.Y., Transgenic CHD1L expression in mouse induces spontaneous tumors, PLoS One. 2009, 4: e6727.

Tam C.N., Cheng M.H., Tsang S.L., Yam G.H.F., Pang C.P. and Sham M.H., Best Poster Award: Unfolded Protein Response Leading to Cataratogenesis in a Microphthalmia Cataract Mouse Mutant., 14th Research Postgraduate Symposium, December 2 & 3, 2009, The University of Hong Kong. 2009.

Tam C.N., Cheng M.H., Tsang S.L., Yam G...H...F..., Pang C...P... and Sham M.H., Best Poster Award: Unfolded protein response leading to cataractogenesis in a microphthalmia cataract mouse mutant., 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium, 15 May 2010. . 2010.

Tam C.N., Cheng M.H., Tsang S.L., Yam G.H.F., Pang C.P. and Sham M.H., Unfolded Protein Response Leading to Cataratogenesis in a Microphthalmia Cataract Mouse Mutant, 14th Research Postgraduate Symposium, December 2 & 3, 2009, The University of Hong Kong. 2009.

Tam C.N., Cheng M.H., Tsang S.L., Yam G...H...F..., Pang C...P... and Sham M.H., Unfolded protein response leading to cataractogenesis in a microphthalmia cataract mouse mutant, 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium, 15 May 2010. . 2010.

Tam C.N., Cheng M.H., Tsang S.L., Yam G.H.F., Pang C.P. and Sham M.H., Unfolded protein response leading to cataractogenesis in a microphthalmia cataract mouse mutant, Mech. Dev. . 2009-09-06, 2009, 126: S128-129.

Researcher : Tsang SW

List of Research Outputs

Chan C.Y., Tsang S.W. and Yao K.M., Secreted PDZ Domain-containing Protein 2 (sPDZD2) Exerts Insulinotropic Effects on INS-1E Cells via a Protein Kinase A-Dependent Mechanism, Japan Society of Developmental Biology 2010 at Kyoto, Japan. 2010.

Tsang S.W., Shao D., Cheah K.S.E., Okuse K., Leung P.S. and Yao K.M., Increased basal insulin secretion in Pdzd2-deficient mice, Mol Cell Endocrinol. 2010, 315(1-2): 263-70.

Researcher : Tsui YP

List of Research Outputs

Shea G.K.H., Tsui Y.P., Chan Y.S. and Shum D.K.Y., Bone marrow-derived Schwann cells achieve fate commitment – a prerequisite for remyelination therapy, Experimental Neurology. 2010, 224: 448-458.

Shea G.K.H., Tsui Y.P., Shum D.K.Y. and Chan Y.S., Fate Commitment in Bone Marrow Stromal Cell-derived Schwann Cells is Dependant on Axonal Contact, 14th Research Postgraduate Symposium, December 2 & 3, 2009, The University of Hong Kong. 2009.

Shea G.K.H., Tsui Y.P., Chan Y.S. and Shum D.K.Y., Fate commitment in bone marrow stromal cells derived Schwann cells is dependant on axonal contact, Proceedings of 32nd Annual Meeting of Japan Neuroscience Society. 2009, P2-d12.

Tsui Y.P., Chan Y.S. and Shum D.K.Y., Bone marrow stromal cell-derived oligodendrocyte precursor cells: A new perspective in CNS myelin repair, 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium, CUHK, Hong Kong, 15 May,. 2010.

Researcher : Tsui YP

List of Research Outputs

Researcher : Tucker W

List of Research Outputs

Tucker W. and Tanner J.A., Aptameric Inhibition of E3 Ubiquitin Ligase WWP1 for Osteoporosis Therapy. , 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium . 2010.

Researcher : Wang C

Project Title:	Imaging Dynamics of Caspase-3 Activation During Developmental Apoptosis by Multiphoton FRET in Living Mouse
Investigator(s):	Wang C, Cheah KSE
Department:	Biochemistry
Source(s) of Funding:	Small Project Funding
Start Date:	01/2009

Abstract:

Apoptosis is an evolutionarily conserved physiological process of cell death involving a cascade of characteristic biochemical and ultrastructural events. In contrast to necrosis, which is a form of traumatic cell death that results from acute cellular injury, apoptosis is an integral part of animal tissue prenatal and postnatal development, and in general, confers advantages during an organism's life cycle. In addition to its importance as a biological phenomenon, defective apoptotic processes have been implicated in an extensive variety of diseases. Diverse stimuli have been demonstrated to be able to active the apoptotic process via two major pathways, the mitochondria pathway or the death receptor pathway, both of which require the activation of a set of cysteine protease termed Caspases and cause the disassembly of the cellular structure and DNA fragmentation which eventually lead to cell death (1). At present, most commonly used assays for apoptosis detection is Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. This assay relies on the presence of nicks in the DNA which can be identified by terminal deoxynucleotidyl transferase, an enzyme that will catalyze the addition of dUTPs that are secondarily labeled with a detective marker (2). However, it may also label the cells undergoing necrosis or cells that have suffered severe DNA damage. Due to the limitation in TUNEL assay, the detection of Caspase-3 activity has been widely used as a compensated tool for detecting apoptosis, since Caspase-3 is the central executioner of apoptosis and is able to cleave and active a number of downstream targets, which trigger a series of apoptotic events (3). However, neither TUNEL assay nor Caspase-3 detection is easy for in situ apoptosis detection or can be used in living animal to study the apoptosis occurrence during tissue development in a spatial and temporal manner. Currently, the advent of genetically targeted biosynthetic sensor technology has significantly increased our ability to study complex signaling events in cells with unprecedented temporal and spatial resolution. Many genetically encoded indicators based on fluorescence resonance energy transfer (FRET) are now available that provide quantitative visualization of cellular alteration in specific subcompartments of cells (4). This project is a collaboration with Professor Donald Chang, (Dept. Biology HKUST) who has developed a sensitive FRET apoptosis biosensor based on the activity of Capase-3 (5). This biosensor contains two green fluorescent protein (GFP) variants cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP), linked by a Caspase-3 specific substrate peptide DEVD. Upon the activation of Caspase-3, these two GFP variants will disassociated, consequently the FRET no longer can be detected. By evaluating the fluorescence alteration, apoptosis occurrence will be easily detected. Currently, they have successfully introduced this biosensor into HeLa cells by both transient and stable gene transfection and demonstrated that this biosensor can be used effectively to measure the activation of caspase-3 during apoptosis in a single living cell (5). Based on that, we aim to extend the application of this FRET Caspase-3 biosensor into living animals, in particular in developing mouse embryos, not just confined to the evaluation of apoptosis in mammalian cells in vitro. To date, no apoptosis specific biosensor mouse model has been reported. Our preliminary work has found expression of this biosensor in transgenic mice at high levels under the control of the β-actin promoter results in prenatal lethality in many transgenic founders, suggesting ubiquitous expression of the Caspase-3 biosensor throughout development was deleterious. Therefore in this project we propose to develop a transgenic conditional apoptosis detection system in which the biosensor is expressed in a spatial manner via Cre/ROSA26-loxP DNA recombination system. The Cre/ROSA26-loxP DNA recombination system is widely used for control of tissue-specific gene expression in mice (6). In this project, the FRET Caspase-3 specific biosensor will be targeted into ubiquitously expressed ROSA26 locus as shown in the attached schematics, separated from ROSA26 promoter by loxP flanked pgk-neo-polyA ‘STOP’ sequences. In the presence of cell type-specific Cre recombinase-mediated excision of the loxP flanked sequence, the biosensor expression is under a spatial control. The main objectives of the present proposal are: (1) Establish FRET based Caspase-3 specific apoptosis biosensor mouse lines via Cre/ROSA26-loxP DNA recombination system. (2) Fully characterize the resultant mouse line to examine the efficiency of the biosensor as an apoptosis indicator (3) Use the biosensor mouse line as a tool to investigate developmental apoptotic events in wild type mice, as well as an apoptosis indicator under disease conditions by breeding with mutant mouse models. References: 1. Samali, A., Zhivotovsky, B., Jones, D., Nagata, S., and Orrenius, S. (1999) Apoptosis: Cell death defined by caspase activation. Cell Death Differ. 6, 495–496. 2. Gavrieli Y, Sherman Y, Ben-Sasson S.A. (1992) Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J Cell Biol. 119, 493-501 3. Cryns, V., and Yuan, J. (1998) Proteases to die for. Genes Dev. 12,1551–1570 4. Breart B, Lemaître F, Celli S, Bousso P. (2008) Two-photon imaging of intratumoral CD8+ T cell cytotoxic activity during adoptive T cell therapy in mice. J Clin Invest. 118, 1390-1397. 5. Luo KQ, Yu VC, Pu Y, Chang DC. (2001) Application of the fluorescence resonance energy transfer method for studying the dynamics of caspase-3 activation during UV-induced apoptosis in living HeLa cells. Biochem Biophys Res Commun. 283, 1054-1060 6. Lewandoski,M. (2001) Conditional control of gene expression in the mouse. Nature Rev. Genet., 2, 743–755 7. Gossen,M. and Bujard,H. (1992) Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc. Natl Acad. Sci. USA, 89, 5547–5551.

List of Research Outputs

Cheah K.S.E., Wang C., Tan Z., Melhado I.G., Niu B., Tsang K.Y., Zhang M...Q..., Ron D... and Chan D., Alleviating ER stress in chondrocytes: survival strategies with double-edged developmental consequences , 43rd Annual Meeting for the Japanese Society of Developmental Biologists Jointly Sponsored by the Asia-Pacific Developmental Biology Network, Kyoto, Japan, 20 June 2010.

Cheah K.S.E., Wang C., Tan Z., Melhado I.G., Niu B., Tsang K.Y., Zhang M...Q..., Ron D. and Chan D., Genetic and genomic analyses of chondrocyte adaptation to ER stress, The Genetics Society Spring Meeting “Mouse Genetics – Think Globally, Act Locally” The Wellcome Trust Conference Centre, Hinxton, UK, 23 April 2010 . 2010.

Researcher : Wang J

List of Research Outputs

Lin R., Lau C.F., Wang J., Ni Y., Fung M.L. and Huang J., Reduced neuronal nitric oxide production in KIF5b-deficiency mice, 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium, CUHK, Hong Kong, 15 May,. 2010.

Researcher : Wang JJ

Project Title:	A systems biology's approach to understand calorie restriction and aging
Investigator(s):	Wang JJ, Huang J, Sham PC, Zhou Z
Department:	Biochemistry
Source(s) of Funding:	Seed Funding Programme for Basic Research
Start Date:	09/2008
Completion Date:	08/2010

Abstract:

Eat less, live longer. Biologists have shown that Calorie Restriction (CR) can extend the life span of various organisms, from bacteria, yeast, to worm and mouse. Mutations that extend lifespan can also reduce the impact of aging-related diseases, including cancer, type II diabetes, cardiovascular disease and neuro-degeneration. However, the underlying protective mechanisms are not well understood. We hypothesize that this is a complex process involving multiple genes and pathways, and there is a common sub-network for these aging-related diseases. In this project, we will employ both computational and experimental tools to unravel the mechanisms of aging, aiming to identify potential therapeutic targets for the prevention and treatment of age-related diseases. Specifically we will 1) integrate genome-wide data from transcriptional profiling, DNA variation, protein-protein interaction, and protein-promoter binding to construct CR related regulatory network; 2) compare this network with the networks of other aging and dietary related diseases to identify candidate genes or gene modules that regulate longevity; 3) to validate these candidates by gene knock-out/knock-down strategy; 4) develop software for network reconstruction, develop database and web resources to facilitate and enhance anti-aging and CR research.

Project Title:	Evolution and functional specialization of alternative promoters in humans
Investigator(s):	Wang JJ, Huang J
Department:	Biochemistry
Source(s) of Funding:	General Research Fund (GRF)
Start Date:	09/2009

Abstract:

1) To compare the evolutions of 5’P and 3’P. We will compare the mutation rates of 5’P and 3’P by four independent measurements, including their information contents, human-mouse conservations, phylogenetic distances, and single nucleotide polymorphisms. We expect all four bioinformatics analyses will test our hypothesis that 5’P and 3’P have different evolution rates. In each test, we will select appropriate human regions as controls[12]. We will compare the evolution of 5’P and 3’P with the controls and test whether APs are under positive/purifying selection; 2) To discover and compare known and novel motifs in 5’P and 3’P. It is important to know which special features exist in the core promoter region that lead to PolII’s selection of a particular alternative promoter for transcription initiation. The core elements are important for PolII assembly and basal level expression of each gene. These core elements include the TATA box, GC box, Inr and DPE[1]. We will compare the occurrences of these core elements in 5’P and 3’P. In addition, we will develop novel approaches to discover new motifs. We expect to identify novel motifs that are unique to 5’P or 3’P, and are responsible for the evolution and functional differences between these two subtypes of promoter; 3) To experimentally compare the roles of 5’P and 3’P in transcription initiation. We will select five representative genes with 5’P and 3’P closest to the consensus to test their roles in transcription initiation. For each gene we will insert its 5’P and 3’P in each orientation and at various spaces into reporter plasmids to study their effects on transcription initiation rates in cells. We will also test the function of our discovered motifs in vitro by mutational analyses. This experimental study will help us to understand the functional roles of different subtypes of promoters on transcription efficiency.

Project Title:	7th Cold Spring Harbor Winter Biotechnology Conference on Systems Biology: Global Regulation of Gene Expression Co-evolution of transcription factor and its binding sites
Investigator(s):	Wang JJ
Department:	Biochemistry
Source(s) of Funding:	URC/CRCG - Conference Grants for Teaching Staff
Start Date:	03/2010
Completion Date:	03/2010

Abstract:

N/A

List of Research Outputs

Wang J.J., A novel profile-HMM to predict microRNA-target interaction, Dept. of Computer and Information Sciences, University of Delaware, Newark, DE, USA. March, 31, 2010.

Wang J.J., A novel profile-HMM to predict microRNAs and their targets simultaneously, Department of Computer Sciences, New Jersey Institute of Technology, Newark, NJ, USA. April 5 , 2010.

Wang J.J., Co-evolution of Transcription Factors and Their Binding Sites, Center for Bioinformatics & Dept. of Genetics, University of Pennsylvania, Philadelphia, PA, USA. March 30 , 2010.

Wang J.J., Yang S., Yalamanchili H.K. and Li X., Co-evolution of transcription factor and its binding sites, Systems Biology: Global Regulation of Gene Expression, CSHL meeting, 23-27, March, 2010.

Wang J.J., PC Member, IEEE International Conference on Bioinformatics & Biomedicine 2009, 1-4 Nov., 2009, Washington D.C. USA.. 2009.

Wang J.J., Prediction of DNA binding sites from transcription factor protein sequence through co-evolution, College of Information Science & Technology, Drexel University, Philadelphia, PA, USA. April 2 , 2010.

Wang J.J., The 8th International Bioinformatics Workshop (IBW2010), 4-6 June, 2010, Wuhan, China, PC member, 2010.

Yang S., Li X.R., Yalamanchili H.K. and Wang J.J., Co-evolution of transcription factors and their binding sites. 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium, Hong Kong. May 15, 2010.

Yang S., Li X.R., Yalamanchili H.K. and Wang J.J., Co-evolution of transcription factors and their binding sites, 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium, CUHK, Hong Kong, 15 May,. 2010.

Yang S., Agrawal K.R., Lam T.W., Sham P.C., Cheah K.S.E. and Wang J.J., Novel Profile Hidden Markov Model to Predict MicroRNAs and their Targets Simutaneously, 14th Research Postgraduate Symposium, The University of Hong Kong, December 2-3. 2009.

Yang S., Agrawal K.R., Lam T.W., Sham P.C., Cheah K.S.E. and Wang J.J., Novel Profile Hidden Markov Model to predict microRNAs in Degenerative Disc Disease. University Grants Council site visit for Area of Excellence Programme "Developmental Genomics & Skeletal Research". January 25, 2010.

Researcher : Wang X

List of Research Outputs

Wang X., Wong E.Y.M., Hui C.C. and Sham M.H., Hoxb1 Cooperates with Gli to Mediate Sonic Hedgehog Signaling in Hindbrain Neurogenesis , 14th Research Postgraduate Symposium, December 2 & 3, 2009, The University of Hong Kong. 2009.

Wang X., Wong E.Y.M., Hui C.C. and Sham M.H., Hoxb1 cooperates with GL1 to mediate sonic hedgehog signaling in hindbrain neural progenitor cell fate determination, International Society for Stem Cell Research, 8th Annual Meeting, Moscone West, San Francisco, CA, USA, June 16-19, 2010.

Wang X., Wong E.Y.M., Hui C.C. and Sham M.H., Hoxb1 cooperates with Gli to activate NKx2.2 expression in the neurogenesis of mammalian hind brain rhombomere 4, 43rd Annual Meeting for the Japanese Society of Developmental Biologists, Jointly Sponsored by the Asia-Pacific Developmental Biology Network, Kyoto, Japan, 20-23 June. 2010.

Wang X., Wong E.Y.M., Hui C.C. and Sham M.H., Hoxb1 cooperates with Gli to activate Nkx2.2 expression in the neurogenesis of mammalian hindbrain rhombomere 4, 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium, CUHK, Hong Kong, 15 May,. 2010.

Wang X., Wong Y.M.E., Hui C.C. and Sham M.H., Hoxb1 cooperates with Gli to active Nkx2.2 expression in the neurogenesis of mammalian hind brain rhombomere 4., 43rd Annual Meeting for the Japanese Society of Developmental Biologists Kyoto, Japan, 20 June 2010 . 2010.

Wang X., ISSCR Young Investigator Travel Award, International Society for Stem Cell Research, 8th Annual Meeting. 2010.

Wang X., Cheung M.C.H. and Chan D., Molecular consequences of Indian hedgehog mutations in chick neural tube, 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium, CUHK, Hong Kong, 15 May,. 2010.

Researcher : Wang X

List of Research Outputs

Wang X., ISSCR Young Investigator Travel Award, International Society for Stem Cell Research, 8th Annual Meeting. 2010.

Researcher : Wang X

List of Research Outputs

Wang X., ISSCR Young Investigator Travel Award, International Society for Stem Cell Research, 8th Annual Meeting. 2010.

Researcher : Wang Y

Project Title:	The potential role of lipocalin-2 as an inflammatory adipokine that links obesity with insulin resistance and metabolic disorders
Investigator(s):	Wang Y, Xu A
Department:	Genome Research Centre
Source(s) of Funding:	General Research Fund (GRF)
Start Date:	01/2008
Completion Date:	06/2010

Abstract:

To use overexpression system to evaluate whether elevation of circulating lipocalin-2 levels can have any effects on systematic insulin sensitivity, inflammation and energy metabolism. As the proposed budget has been cut, we will remove the second part of the experiment on using the euglycemic-hyperinsulinemic clamp for evaluating individual tissue insulin sensitivities. Nevertheless, the overall goal of this part of work will not be affected as the other measurements on insulin sensitivity (GTT and ITT), metabolic parameters (lipid, insulin and glucose levels) as well as the inflammatory markers will still be carried on for this objective and can help to elucidate the systematic effects of lipocalin 2; to focus on evaluating whether lipocalin2 deficiency can prevent the development of insulin resistance using lipocalin 2 knockout mice challenged with high fat diet and genetic obesity (db/db background). On the other hand, we will remove the neutralization experiment using lipocalin-2 antibody in order to save the cost. Again, this revised plan will not affect the overall objectives as both knocking-down approaches will achieve similar effects; to agree with the reviewer 1's suggestion that AMPK experiment is not necessary. In addtion, since we will be focusing on inflammatory and insulin-signalling pathways, the metabolic pathway Oligo GEArray analysis will not be performed. In the mean time, the investigations on the target tissues, including liver, adipose tissue and skeletal muscle will continue to be carried on. This more focused experimental plan will help the smooth running of the project by fitting in with the current budget and will not sacrifice general project objectives.

Project Title:	Cross-talk between SIRT1 and insulin signaling pathways: Potential roles in regulating systemic insulin sensitivity and energy metabolism
Investigator(s):	Wang Y, Xu A
Department:	Genome Research Centre
Source(s) of Funding:	Seed Funding Programme for Basic Research
Start Date:	06/2008
Completion Date:	12/2009

Abstract:

Sirtuins are a family of NAD+-dependent protein deacetylases that regulate cellular functions through deacetylation of a wide range of signaling molecules, transcription factors, histones and enzymes etc. Sir2 (silent information regulator 2), the first gene discovered in this family, was originally shown to be involved in transcriptional silencing at cell-mating type loci and telomeres in yeast, and suppression of recombination at yeast ribosomal DNA (rDNA), through deacetylation of the epsilon-amino groups of lysines in the amino-terminal domains of histones [1-3]. Yeast sirtuins (Sir1-4)-mediated silencing contribute to the fundamental cellular processes such as proper cell cycle progression, radiation resistance, and genomic stability etc [4]. After years of intense studies, it is now clear that sirtuins are phylogenetically conserved from bacteria to humans and regulate cell functions far beyond gene silencing. The anti-aging effects of Sir2 was firstly demonstrated by Kaeberlein et al, who showed that in S. cerevisiae, integration of extra copies of Sir2 extended lifespan up to 30% [5]. Similar effects of Sir2 were subsequently observed in Caenorhabditis elegans and Drosophila melanogaster [6-9]. Overexpression of Sir-2.1 increased lifespan up to 50% in C. elegans. In Drosophila, an extra copy of Sir2 gene extended lifespan in female and male by 29% and 18% respectively. Seven human homologues of sirtuins, SIRT1-7, have been characterized to share the catalytic domain with Sir2 [10-12]. Like other family members of sirtuins, SIRT1-3 and 5 show NAD+-dependent protein deacetylase activities, whereas SIRT4 and 6 have been found to possess mono-ADP-ribosyl transferase activities [13-17]. Recent research indicates that through modulating the acetylation and deacetylation of various target proteins, sirtuins can elicit their diversified functions in cell type-specific manners, which have pathophysiological implications in cancer, obesity, inflammation and neurodegenerative diseases. The requirement of NAD+ as a co-substrate suggests that sirtuins might act as sensors of cellular energy and redox states and could be regulated by the cellular metabolic status. Indeed, yeast Sir2 and the mammalian homologue SIRT1 can be upregulated by calorie restriction, which promote survival in organisms ranging from yeast to rodents and primates [18, 19]. Despite that the roles of SIRT1 in mammalian aging have not been fully characterized, mounting evidences suggest that SIRT1 could be an important regulator in systemic energy metabolism and metabolic syndrome, and that the anti-aging effects of SIRT1 might be related to its metabolic regulations. In mice, the beneficial metabolic profiles associated with calorie restrictions, including improved glucose tolerance (lower blood glucose and insulin levels), decreased LDL cholesterol and triacylglycerol and increased HDL cholesterol etc, are at least partially attributed to the elevated SIRT1 expression levels [18, 20]. Transgenic mice overexpressing SIRT1 are leaner, more metabolically active and glucose tolerant, and display decreased circulating levels of lipid, glucose and insulin [21]. SIRT1 knockout mice show lower blood glucose and increased glucose tolerance compared with the wild-type mice [22]. Resveratrol, a polyphenol found in red wine that contributes to the “French paradox”, a phenomenon of lower incidence of metabolic diseases despite their high saturated fat diet, might elicit its metabolic regulatory effects through activating SIRT1 [23-26]. The metabolic regulatory effects of SIRT1 on individual tissue have also been reported. SIRT1 inhibits adipogenesis in white adipose tissue by repressing activity of the proadipogenic nuclear receptor, peroxisome proliferator-activated receptor γ (PPARγ) [27]. SIRT1 enhances insulin secretions from pancreatic β cells by regulating UCP2 expressions [22, 28]. SIRT1 deacetylates and activates the transcriptional coactivator PGC-1α to increase gluconeogenesis in the liver [29, 30]. Notably, many of these metabolic functions of SIRT1 are observed under fasting status. Taken together, these results suggest that in mammalian system, SIRT1 possesses a broader range of metabolic regulatory functions in a tissue-specific manner, which might play important roles in maintaining energy homeostasis under different nutrient conditions and eventually executing a programme for extended lifespan. Despite these advances, the detailed signaling pathways and molecular mechanisms responsible for SIRT1-regulated cellular metabolism are far from clear. Sirt1 can regulate several transcription factors that govern metabolism, including PPARγ, PPARγ-coactivator 1α, and forkhead-box transcription factors (FOXOs), through direct interactions or modulating their acetylation/deacetylation status [31]. However, many of the metabolic effects mediated by SIRT1 could not be simply explained by activations of these transcription factors. The possible involvement of SIRT1 in insulin-signaling pathways has been suggested by the following evidences: Firstly, the insulin-IGF-I signaling pathway is nutrient activated, and decreased signaling through this pathway increases life span in C. elegans [32, 33]. and in mice [34]; Secondly, both calorie restriction and SIRT1 overexpression cause the reduced fat masses in mice, antagonizing the effects of insulin-induced fat storage and fat cell differentiation [27]; Thirdly, in the liver, overexpression of SIRT1 elicits catalytic activities, stimulates gluconeogenesis and fatty acid oxidation, and operates against the hepatic insulin response pathway, which stores glucose and represses gluconeogenesis [29, 35, 36]; Fourthly, SIRT1 and insulin possess opposing effects on the functions of PGC-1α and FOXO, two transcription factors involved in regulating metabolism and aging [37-40]; Furthermore, resveratrol, a well-known activator of SIRT1, inhibits the activity of PI3K and its downstream targets in human primary myotubes and muscle-derived cell lines, as well as in primary hepatocytes and liver-derived cell lines [41-43]. Collectively, these data suggest that SIRT1 might exert its metabolic functions through modulating insulin signaling activities. This project is thus designed to dissect the detailed cross-talks between SIRT1 and the insulin signaling pathways using both in vitro and in vivo approaches. The results are expected to shed important insights on the mechanisms that mediate the diversified metabolic functions of sirtuins. Specific objectives are: 1. To investigate whether overexpression of SIRT1 can antagonize insulin-evoked signaling pathways in several insulin-responsive cell lines (HepG2, C2C12 myocytes and 3T3-L1 adipocytes). 2. To evaluate whether or not SIRT1 physically interacts with the signaling molecules downstream of insulin receptor and modulate their acetylation/deacetylation status. 3. To elucidate whether adipose tissue-specific overexpression of SIRT1 could affect adiposity, systemic insulin sensitivity and energy metabolism in mice.

Project Title:	Molecular mechanisms underlying the hepato-protective functions of the fat cell-derived hormone adiponectin: potential roles of uncoupling protein 2
Investigator(s):	Wang Y, Xu A
Department:	Biochemistry
Source(s) of Funding:	General Research Fund (GRF)
Start Date:	01/2009

Abstract:

(1) To evaluate whether over-expression of UCP2 could alleviate the liver injuries associated with adiponectin deficiency and whether UCP2 ablation could abolish the hepatoprotective functions of adiponectin in mice: (2) To elucidate the detailed molecular and cellular mechanisms whereby UCP2 mediates the effects of adiponectin on inhibition of ROS production, inflammation and apoptosis in both primary hepatocytes and Kupffer cells; (3) To delineate the potential molecular and signaling pathways underlying the stimulatory effects of adiponectin on UCP2 expression.

Project Title:	Elucidation of the molecular mechanisms underlying endothelial aging using integrated proteomic profiling approaches
Investigator(s):	Wang Y, Vanhoutte PMGR
Department:	Pharmacology
Source(s) of Funding:	Seed Funding Programme for Basic Research
Start Date:	06/2009
Completion Date:	09/2010

Abstract:

Aging is a physiological process closely associated with the development of cardiovascular mobility and mortality independent of known cardiovascular risk factors1. Aging-related changes in the blood vessel include decreased antithrombogenic property of the endothelium, increased inflammation, impaired angiogenesis, reduced endothelium-dependent vasodilatations, as well as elevated expression of adhesion and proinflammatory/prothrombogenic molecules2. Endothelial cell senescence plays an important role in causing these aging-related vascular functional changes3. Cellular senescence originally referred to a limited ability of human cells to divide when cultured in vitro, a phenomenon accompanied by a specific set of phenotypic changes in morphology, gene expression and function4. It is now accepted that cellular senescence is a natural biological process. Its role in vivo is not unclear, and the specific molecular mechanisms underlying biological aging remain largely uncharacterized. Senescent endothelial cells cease to proliferate and do not respond to mitogenic stimuli. They lose their function and original morphology, but acquire a flattened cytoplasm (“fried egg” appearance), and can be frequently found on the surface of atherosclerotic plaque5. They show increased beta-galactosidase activity and impaired in vitro growth properties. The production of nitric oxide and eNOS activity are reduced in senescent human endothelial cells. Stimulation with proinflammatory cytokines exacerbates monocyte-endothelial cell interactions more profoundly in these cells. Thus, targeting endothelial cell senescence represents a promising therapeutic strategy for the treatment of atherosclerosis6. One popular explanation for senescence is the telomere hypothesis. Telomeres play a critical role in vascular cell senescence7-9. They are nonnucleosomal DNA-protein complexes at the end of chromosomes and serve as protective caps. During cell division, the extreme termini of chromosomes are not duplicated completely, which results in successive shortening of telomeres. The onset of senescence will be triggered by extremely short telomeres. The enzyme telomerase reverse transcriptase (TERT) and associated proteins are responsible for adding telomeres onto chromosome ends with its RNA moiety as a template. Both the validity of telomere length and the dynamic telomere components are critically involved in determining cell viabilities or aging10. Telomere homeostasis is regulated through multiple mechanisms, including protein composition, telomere length, and telomerase activity levels. In primary endothelial cell cultures, limited proliferative capacity correlates with telomere attrition and forced expression of human TERT results in extended cell life span or immortalization11. A direct correlation exists between TERT expression and neovascularization12. Endothelial cells from human abdominal aortae display age-dependent telomere shortening13. Correlations exist between short telomeres and hypertension, cardiovascular diseases, and myocardial infarction are consistently found 14. However, the detailed molecular mechanisms underlying telomere shortening and telomerase inactivation remain largely uncharacterized. Our laboratory has established a reliable primary cell culture model for evaluating endothelial function and senescence15. Endothelial cells are harvested from the coronary arteries of female pig hearts and cultured with the medium changed every 48 hours. Cells are detached with trypsin-EDTA and further passaged at a ratio of 1:3 at regular intervals (once per week) for 4 weeks. Primary porcine endothelial cells (PPECs) have a limited life span in culture. After four to five passages, they tend to de-differentiate and eventually reach senescence16. The cumulative population doubling is 19.18 from passage one to four, at which the cells show senescence and decreased NO production17. Microarray analysis revealed that their mRNA expression pattern resembles those observed in regenerated endothelium15, which has been proliferating in vivo for 4 weeks after balloon injury. The reduced proliferative capacity, the functional deterioration as well as the morphological changes from “cobblestone-like” young endothelial cells to the enlarged and flattened senescent endothelial cells are comparable between the two forms of senescence occurring in vivo and in vitro15, 17-20. More recent results demonstrate that the progressively decreasing telomerase activity is closely associated with the occurrence of senescence in PPECs (Figure 1), and that replenishment of an anti-aging protein SIRT1 restores the diminished telomerase activities (data not shown). Therefore, in order to further understand the underlying mechanisms of endothelial senescence, we plan to use proteomics-based approaches to systematically characterize the components of telomere complex and their dynamic changes during the aging process. The specific objectives include: 1. To purify and identify the components of telomerase complexes in normal and senescent PPECs using biochemical separations and Multidimensional Protein Identification Technology (MudPIT). 2. To characterize the detailed post-translational modifications occurring on TERT that may be associated with cellular senescence and contribute to the decreased telomerase activity. 3. To profile the differentially expressed nuclear proteins in PPECs undergoing senescence using isobaric tag peptide labeling mass spectrometry technology.

Project Title:	Discovery of novel inhibitors targeting lipocalin-2 for the treatment of obesity-related diabetes and cardiovascular diseases
Investigator(s):	Wang Y, Vanhoutte PMGR, Xu A
Department:	Pharmacology
Source(s) of Funding:	Seed Funding Programme for Applied Research
Start Date:	06/2010

Abstract:

Type 2 Diabetes Mellitus (T2DM) and cardiovascular diseases (CVD) are two major causes of mortality and morbidity in the ageing population. Obesity is the most common risk factor for these inter-related metabolic and cardiovascular disorders. Chronic inflammation of the adipose tissue and dysregulated production of adipokines are the key mechanisms linking obesity to its associated pathologies (1, 2). The pro-inflammatory adipokines, such as leptin, resistin, retinol binding protein 4 and visfatin, act either in an autocrine manner to perpetuate local inflammation, or in an endocrine manner to induce systemic metabolic and vascular dysfunctions (3). Targeting these adipokines represents promising strategies for the treatment of obesity-associated medical complications. Several lipocalins produced in adipose tissue have been implicated in obesity-related metabolic syndrome and cardiovascular dysfunctions. For example, adipose tissue expression of retinoid binding protein-4 (RBP4) and the serum levels of this protein are elevated in insulin-resistant mice and in humans with obesity and T2DM, but normalized by the insulin-sensitizing drug rosiglitazone (4). Transgenic over-expression of human RBP4 or injection of recombinant RBP4 in normal mice causes insulin resistance (5). Conversely, genetic deletion of RBP4 enhances insulin sensitivity. Another adipocyte-produced lipocalin, adipocyte fatty acid binding protein (A-FABP) also plays important roles in integrating systemic energy homeostasis, insulin sensitivity and inflammation (6). Targeted disruption of the A-FABP gene provides significant protection against both dietary and genetic obesity-associated insulin resistance, T2DM and fatty liver diseases, and also leads to marked alleviation of inflammation and atherosclerosis associated with ApoE deficient mice (6, 7). Orally active small-molecule inhibitor of AFABP is an effective therapeutic agent against severe atherosclerosis and type 2 diabetes in mouse models (8). Lipocalin-2, a 25-kDa secretory glycoprotein originally purified from human neutrophils, is highly expressed in adipose tissue (9-13). This protein structurally belongs to the lipocalin superfamily, with a characteristic cavity for binding small lipophilic substances (14). Recent studies from our laboratory and others have demonstrated a pivotal role of lipocalin-2 in the pathogenesis of obesity-related diabetes in both human and animal models (10-12). Lipocalin-2 expression in “inflamed” adipose tissue and plasma concentrations of this protein are markedly increased in obese/diabetic mice and humans, and that the augmented expression can be reversed by rosiglitazone, an insulin-sensitizing and anti-diabetic drug. Mice without lipocalin-2 are protected from ageing- and obesity-associated insulin resistance (15). Compared with their wild type littermates, obese lipocalin-2 knockout mice (Lcn2-KO) show significantly decreased fasting glucose and insulin levels and improved insulin sensitivity. Overexpression of lipocalin-2 increases fasting glucose and insulin levels, and reduces insulin sensitivity in both wild type and Lcn2-KO mice. Obesity and diabetes are major risk factors for endothelial dysfunction, an early manifestation of vascular disorders (16). Under these conditions, endothelial cells can induce contractions of the underlying vascular smooth muscle by generating endothelium-dependent contracting factor (EDCF). EDCF can be enhanced by both high fat diet feeding and aging in mice. Our more recent work suggests that acetylcholine-induced EDCF-mediated responses are abolished in lipocalin-2 deficient mice. The contractions were inhibited by indomethacin (non-selective COX inhibitor), SC560 (COX-1 inhibitor) and S18886 (TP receptor antagonist), but not NS398 (COX-2 inhibitor), suggesting the involvement of COX-1. Lipocalin-2 treatment in cultures of endothelial cells promotes COX-1 expression trough a ROS-dependent mechanism. Additionally, in both aging and high fat diet conditions, LCN2-KO mice exhibit an increased aortic sensitivity to insulin-induced vasodilatation of aorta rings, which is accompanied by an enhanced insulin-stimulated eNOS phosphorylation. These evidence strongly support lipocalin-2 to be a causal factor in the development of insulin resistance, metabolic and vascular abnormalities. Hence, pharmacological agents that inhibit lipocalin-2 activity may offer therapeutic opportunities for obesity-associated metabolic, cardiovascular and inflammatory diseases. We have already filed a patent through HKU versitech to claim the use of lipocalin-2 as a therapeutic target to design the drugs for treatment of obesity and diabetes (US patent appl No: 20080095782). This application aims to collaborate with Guangzhou Institute of Biomedicine & Health, Chinese Academy of Sciences to search for potent chemical inhibitors of lipocalin-2 as lead compounds, and to test their bioactivities in animal models. The data will be used to support the application for Guangdong-Hong Kong Technology Cooperation Funding Scheme (TCFS) of Innovation & Technology Funding (ITF) in the future. Specific objectives are: 1. Identification of the endogenous ligands binding to lipocalin-2 for structural-based drug design. 2. High-throughput screening of lipocalin-2 inhibitor compounds using fluorescence probe-based assays and affinity chromatography coupled to mass spectrometry analysis. 3. Cellular and animal-based assay for functional characterization and validation of lipocalin-2 inhibitors.

Project Title:	Lipocalin-2 and myocardial remodeling in response to ischemia-reperfusion injury
Investigator(s):	Wang Y, Vanhoutte PMGR, Xu A
Department:	Pharmacology
Source(s) of Funding:	Seed Funding Programme for Basic Research
Start Date:	06/2010

Abstract:

Obesity and the associated disorders [insulin resistance, hypertension, hyperlipidemia and type 2 diabetes mellitus (T2DM)] have a major impact on the incidence, severity, and outcome of ischemic heart disease (1). In the Framingham heart study, greater body mass index (BMI) is associated with an increased risk of heart failure in both men and women (2). The risk can be graded across categories of increasing BMI. Moreover, higher mortality rates as well as higher re-infarction and heart failure rates are found in diabetic patients, both during the acute phase and in extended postinfarction periods. While it is widely accepted that obesity increases the risk of developing heart disease, a number of reports also suggest a statistically significant survival benefit in obese patients with cardiovascular diseases (3). This phenomenon, referred as “obesity paradox”, has been described in patients with heart failure, particularly systolic heart failure (4), in cohorts with hypertension (5), coronary heart disease (6) and peripheral arterial disease (7). Importantly, evidence from clinical studies suggest that drugs for improving blood pressure and glucose metabolism may harm myocardial performance (8). Obesity is associated with structural changes in the heart. Pathologic cardiac remodeling, such as left ventricular (LV) hypertrophy, left atrial (LA) enlargement, and subclinical impairment of LV systolic and diastolic functions, are the precursors to overt cardiac dysfunction and heart failure (9). Increased cardiac mass has been postulated to result from increased epicardial fat and fatty infiltration of the myocardium (10). Increased accumulation of intramyocellular triglycerides and lipid metabolites in the heart has been found in the hearts of genetic obese animals, including ob/ob and db/db mice, Zucker rats, and those fed with high fat diet (9, 11, 12). Alterations in myocardial fatty acid metabolism and efficiency can cause decreased cardiac performance. Obese subjects, particularly those with insulin resistance, show increased myocardial fatty acid uptake and utilization (13), and subclinical contractile abnormalities (14). Animal studies using in vivo models, isolated perfused hearts, or in vitro cardiomyocyte cultures suggest that many of the changes in cardiac function are dependent on effects that may be secondary to obesity and altered glucose/lipid metabolism. Subtle changes in cardiac function can be observed in isolated hearts at time points when significant changes are not apparent when evaluated in vivo. For example, mildly impaired systolic function in vivo is absent in younger obese animals (<12 weeks of age), and become evident between 12 and 20 weeks of age, when evaluating by echocardiography. However, in isolated working hearts, significant reduction in cardiac power can be detected despite preserved in vivo cardiac function in animals before developing obesity and diabetes (15). Adipose tissue, once considered simply a lipid storage depot, is now known to function as a secretory organ producing a variety of bioactive molecules referred to as adipokines (16). Adipokines are believed to directly or indirectly affect the pathophysiology of various obesity-linked disorders and biological processes. Dysregulated adipokine production has been implicated in obesity-related cardiomyopathy. A number of these adipokines, such as leptin, adiponectin and apelin, elicit endocrine and paracrine effects on regulating cardiac functions (17, 18). For example, plasma adiponectin levels are inversely associated with the risk of myocardial infarction (19), and rapidly decline after acute myocardial infarction (20). Mice without adiponectin undergo worse myocardial ischemia-reperfusion injury than wide type control mice (21). Adiponectin mediates antihypertrophic effects in the heart in part through activation of AMPK signaling (22). Leptin concentrations are inversely correlated with LV mass, LV wall thickness, and left atrial size (23). Cross-sectional studies suggest a cardioprotective influence of leptin on LV remodeling. In fact, the temporal nature of changes in cardiac structure and function suggest that the “obesity paradox” phenomena may in part be explained by the dynamic actions of adipokines on the myocardium. Lipocalin-2 is an adipokine up-regulated in obese subjects (24). Its plasma levels are closely correlated with various metabolic and inflammatory parameters. Mice with deletion of the lipocalin-2 gene (Lcn2-KO) show improved systemic insulin sensitivity, decreased inflammatory cytokine production and attenuated inflammation in adipose tissue. Replenishment with lipocalin-2 in Lcn2-KO mice increases circulating blood glucose and insulin levels, and causes insulin resistance, suggesting that this adipokine is causally involved in the development of obesity-associated insulin resistance and metabolic abnormalities (25). There are a number of studies concerning the role of lipocalin-2 in the cardiovascular system. Its expression is significantly augmented in patients with coronary heart disease and independently associated with systolic arterial blood pressure, insulin resistance and HDL cholesterol (26). Lipocalin-2 levels are increased in atherosclerotic plaques and myocardial infarction (27). It may mediate the innate immune responses in the pathogenesis of heart failure (28-31). Recently, we have used the Langendorff-perfusion system to evaluate the heart functions of mice without lipocalin-2. Our results demonstrate that Lcn2-KO mice are protected from dietary obesity-induced impairment of heart functions (Figure 1). After 30 min of global ischemia and 60 min of reperfusion, Lcn2-KO mice show significantly improved recovery of LV contractility and decreased myocardial cell apoptosis compared to wide type mice (Figure 1, A and B). Moreover, histological analysis reveals that lipocalin-2 deficiency may modulate the myocardial structures (Figure 1C). These observations support a potential role of lipocalin-2 in the pathogenesis of obesity-related cardiac disorders. Therefore, this study aims to perform mechanistic analyses at cellular, molecular and systematic levels for understanding the pathophysiological roles of lipocalin-2 in myocardial remodeling in response to ischemia-reperfusion (I/R) injury in vivo. The specific objectives are: 1. To investigate whether the expression levels of lipocalin-2 are altered in the mice heart tissues undergoing myocardial I/R injury, and to analyze whether lipocalin-2 deficiency or replacement will affect the cardiac remodeling process in mice. 2. To examine whether lipocalin-2 deficiency affects the fat infiltration/ accumulation and the lipid profiles in the mice heart tissues, and to evaluate whether lipocalin-2 elicits direct effects on cardiomyocyte metabolism and function. 3. To identify possible mechanisms by which lipocalin-2 regulates cardiac remodeling, cardiomyocyte metabolism and function.

Researcher : Wang Y

List of Research Outputs

Wang Y., Lu L., Wu M.H., Sham M.H., Chan D., Cheah K.S.E. and Cheung M.C.H., In vitro generation of Osteo-chondroprogenitors, 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium, CUHK, Hong Kong, 15 May,. 2010.

Researcher : Wang Z

List of Research Outputs

Cui J., Wang Z., Cheng Q.N., Lin R., Zhang X., Leung P.S., Copeland N.G., Jenkins N.A., Yao K.M. and Huang J., Targeted Inactivation of KIF5B in Pancreatic Leads to Insulin Secretory Deficiency and Increased b-Cells Mass in vivo, 14th Research Postgraduate Symposium, December 2 & 3, 2009, The University of Hong Kong. 2009.

Liu C., Wang Z., Huen M.S.Y., Lu L., Liu D. and Huang J., Cell death caused by single-stranded oligodeoxynucleotides mediated targeted genomic sequence modification, Oligonucleotides. 2009, 19(3): 281-286.

Researcher : Wang Z

List of Research Outputs

Researcher : Wong CM

Project Title:	Transcription termination in Saccharomyces cerevisiae
Investigator(s):	Wong CM, Jin D
Department:	Biochemistry
Source(s) of Funding:	Small Project Funding
Start Date:	09/2008
Completion Date:	02/2010

Abstract:

Project objectives: A. Regulation of transcription termination via post-translation modification of Npl3p B. The role of exosome/TRAMP in transcription termination For part A, the major hypothesis is transcription termination sites could be selected via post-modification of the anti-terminator, Npl3p. The recruitment of Npl3p to termination sites can be modulated by post-translational modifications which will affect its localization, RNA-binding affinity and/or protein-protein interaction. Npl3p is a SR-like hnRNP (heterogeneous nuclear ribonucleoprotein) with documented roles in mRNA export, ribosome biogenesis and transcription termination. According to the current model, Npl3p prevents transcription termination by competing with the cleavage factors Hrp1p and Rna15p for binding to nascent transcripts. Npl3p has a higher affinity to RNA than do Hrp1p and Rna15p. This property is important since Npl3p functions as a physical barrier hindering Hrp1p and Rna15p from binding to nascent transcript. However, Hrp1p and Rna15p show higher affinity to AU-rich sequence (strong termination site signal) than Npl3p. Npl3p will hence be displaced by Hrp1p and Rna15p. As a result, Hrp1p and Rna15p induce termination by cleaving the nascent transcript. In fact, signals in the DNA that define termination are very poorly defined, and transcription termination sites can be varied. Interestingly, the RNA-binding affinity and localization of Npl3p can be modulated by posttranslational modifications, including methylation (by Hmt1p) and phosphorylation (mainly by Sky1p). In this part, we are going to investigate the effect of these Npl3p modifications on transcription termination. For part B, we will focus on the investigation of exosome/TRAMP in transcription. We are going to perform systematic screening the components of exosome/TRAMP for their novel function in transcription elongation (both elongation rate and processivity), termination and RNA processing (translocation and degradation). Aberrant transcripts are retained in the nucleus and subjected to degradation by exosome/TRAMP. This transcript degradation mechanism prevents potential toxic truncated proteins from being produced. Exosome is a complex of 3′–5′ exonucleases and it degrades RNA in RNA-protein complexes. TRAMP functions as exosome cofactor, which enhances the activity of exosome in vitro. However, when and how the components of exosome/TRAMP complex are recruited during transcription so as to monitor the quality of nascent transcripts are poorly understood. In this part, we will focus on the investigation of exosome/TRAMP in transcription.

Project Title:	2009 CSHL Meeting on Mechamisms of Eukaryotic Transcription Yeast arginine methyltransferase Hmt1p is required for transcription elongation and termination
Investigator(s):	Wong CM
Department:	Biochemistry
Source(s) of Funding:	URC/CRCG - Conference Grants for Teaching Staff
Start Date:	08/2009
Completion Date:	08/2009

Abstract:

N/A

List of Research Outputs

Tang V.H.M., Siu K.L., Wong C.M. and Jin D., Loss of Yeast Peroxiredoxin Tsa1p Induces Genome Instability through Activation of the DNA Damage Checkpoint and Elevation of dNTP Levels, In: Orna Cohen-Fix, PLoS Genetics. 2009, 5(10): e1000697.

Wong C.M., Tang V.H.M., Kong K.Y., Wong G.W., Qiu H., Jin D. and Hinnebusch A.G., Yeast arginine methyltransferase Hmt1p regulates transcription elongation and termination by methylating Npl3p, Nucleic Acids Research. 2010, 38(7): 2217-2228.

Researcher : Wong EYM

List of Research Outputs

Chu K.H., Zhang M., Wong E.Y.M., Szeto Y.Y., Chan Y.S., Cheah K.S.E. and Sham M.H., Best Poster Award: Sox10 mutation affects cochleo-vestibular ganglion gliogenesis, 43rd Annual Meeting for the Japanese Society of Developmental Biologists, Kyoto, Japan, 20-23 June, 2010.. 2010.

Chu K.H., Zhang M., Wong E.Y.M., Szeto Y.Y., Chan Y.S., Cheah K.S.E. and Sham M.H., Best Poster Award: Sox10 mutation affects gliogenesis of the cochleo-vestibular ganglion, 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium, 15 May 2010. 2010.

Chu K.H., Zhang M., Wong E.Y.M., Szeto Y.Y., Chan Y.S., Cheah K.S.E. and Sham M.H., Sox10 mutation affects cochleo-vestibular ganglion gliogenesis, 43rd Annual Meeting for the Japanese Society of Developmental Biologists, Kyoto, Japan, 20-23 June. 2010.

Chu K.H., Zhang M., Wong E.Y.M., Szeto Y.Y., Chan Y.S., Cheah K.S.E. and Sham M.H., Sox10 mutation affects gliogenesis of the cochleo-vestibular ganglion, 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium, 15 May 2010.

Wang X., Wong E.Y.M., Hui C.C. and Sham M.H., Hoxb1 Cooperates with Gli to Mediate Sonic Hedgehog Signaling in Hindbrain Neurogenesis , 14th Research Postgraduate Symposium, December 2 & 3, 2009, The University of Hong Kong. 2009.

Wang X., Wong E.Y.M., Hui C.C. and Sham M.H., Hoxb1 cooperates with GL1 to mediate sonic hedgehog signaling in hindbrain neural progenitor cell fate determination, International Society for Stem Cell Research, 8th Annual Meeting, Moscone West, San Francisco, CA, USA, June 16-19, 2010.

Wang X., Wong E.Y.M., Hui C.C. and Sham M.H., Hoxb1 cooperates with Gli to activate NKx2.2 expression in the neurogenesis of mammalian hind brain rhombomere 4, 43rd Annual Meeting for the Japanese Society of Developmental Biologists, Jointly Sponsored by the Asia-Pacific Developmental Biology Network, Kyoto, Japan, 20-23 June. 2010.

Wang X., Wong E.Y.M., Hui C.C. and Sham M.H., Hoxb1 cooperates with Gli to activate Nkx2.2 expression in the neurogenesis of mammalian hindbrain rhombomere 4, 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium, CUHK, Hong Kong, 15 May,. 2010.

Researcher : Wong NS

Project Title:	Kappa-opioid receptor mediated cellular stress response: the role of Inositol 1,4,5-trisphosphate and diacylglycerol
Investigator(s):	Wong NS, Yao KM
Department:	Biochemistry
Source(s) of Funding:	General Research Fund (GRF)
Start Date:	10/2002

Abstract:

The Phospholipase-C (PLC)/inositol-lipid signaling mechanism is itself of widespread importance in cellular regulation and is coupled to all the three major subtypes of opioid receptors (OR), but how this signaling pathway mediates OR-simulated cellular responses is scarcely known. The objective of this project is to investigate the importance of the PLC/inositol-lipid pathway in KappaOr-induced stress responses.

Project Title:	*Redox-mediated posttranscriptional regulation of CHOP* expression: its implication in CHOP-mediated pathologies**
Investigator(s):	Wong NS, Wang Y
Department:	Biochemistry
Source(s) of Funding:	General Research Fund (GRF)
Start Date:	01/2007

Abstract:

(1) Characterization of the 625-688 (-60bp) region and the ARE; (2) regulation of CHOP mRNA stability by known RNA-binding protein; (3) identification of trans-acting RNA binding proteins.

Project Title:	The investigation of the relation between oxidative stress and ER-stress
Investigator(s):	Wong NS
Department:	Biochemistry
Source(s) of Funding:	Seed Funding Programme for Basic Research
Start Date:	04/2008
Completion Date:	03/2010

Abstract:

The endoplasmic reticulum (ER) of a cell is an elaborate membranous structure where the synthesis of most of the cellular proteins takes place. Nascent polypeptides synthesized at the ribosomes are translocated into the lumen of the ER to undergo posttranslational modifications as well as proper folding into functional proteins. The folding of polypeptides requires also the assistance of chaperone proteins and the formation of disulphide bonds. The latter process is catalysed by protein disulphide isomerase (PDI). The capacity of the ER to produce properly folded proteins is determined amongst many other factors, the availability/abundance of the chaperone proteins, the level of PDI activity, and a relatively hyperoxidising environment within the ER that is optimized for disulphide bond formation to occur. ER-stress develops when the protein folding capacity of the ER can no longer cope with the cell’s demand for protein synthesis. Such is the case when professional secretory cells received physiological cues that call for an increased output of a particular protein, for example, insulin (secreted by the pancreatic β-cell) and IgG (secreted by plasma cells). ER-stress arising from pathological causes is exemplified by viral hijacking of normal cells that are subsequently directed by the virus to produce huge quantities of viral proteins. Alternatively, ER-stress can be produced by the abolition of conditions optimised for protein folding in the ER, as illustrated by the inhibition of Ca2+ transport across the ER-membrane (exerted by thapsigargin), and the inhibition of protein glycosylation (exerted by tunicamycin). During ER-stress, many of the newly polypeptides remained partially or improperly unfolded. If such protein-like species were allowed to accumulate, precipitation will eventually occur causing damage to ER through a process described as proteotoxicity. Purpose of the project The purpose of the present project is to understand further how ER-stress may be produced in cells. Previous studies revealed that ER-stress is closely associated with oxidative stress. How oxidative-stress may arise subsequent to ER-stress is currently unknown. However, the presence of oxidative stress in cells has been known to promote/aggravate ER-stress. This is because during protein-folding, the redox environment of the ER has to be controlled stringently so that the oxidation of pairs of thiol groups to form disulphide bonds and the reverse of this reaction, can take place continuously in the folding polypeptide - a process known as reshuffling of disulphide bonds and is essential for protein folding. Oxidative stress destroys the redox environment in the ER and thus prevents disulphide reshuffling. Consequently, in the presence of oxidative stress the correct folding of polypeptides will be interupted, and ER-stress is produced as a result. Much less is known concerning how ER-stress will produce oxidative stress. Cells respond to ER-stress with the activation of the unfolded protein response (UPR) - an elaborate and evolutionarily conserved mechanism that is intended to enable cells to adapt and hence to survive ER-stress. UPR is activated by three signaling pathways that are able to sense ER-stress by the three protein sensors (PERK, Ire1 and ATF6) located in the ER. The activation of UPR enables cells to reduce the protein load of the ER, to increase the folding capacity of the ER, and to activate the degradation of incorrectly folded proteins. It has been suggested that oxidative stress produced subsequent to ER-stress is due to UPR. Key issues Since oxidative stress is able to create ER-stress and the latter can in turn produce oxidative stress, it is speculated that a positive feedback loop could form to amplify the effect of any stimulus of ER-stress production. Any ER-stress produced in cells will then be exaggerated by such a putative positive feedback effect. The implication of such a positive feedback mechanism is profound. It may be beneficial to normal cells since it will enhance the sensitivity towards ER-stress and reduce the ER-stress threshold for UPR activation. However, the cell may also become less tolerant to high ER-stress eventually leading to cellular demise. In abnormal (cancer) cells, such a feedback mechanism may enhance the toxic effect of anticancer drugs that is mediated by ER-stress. The investigation of the existence of such a positive feedback mechanism and its role in cell physiology and pathology will provide further insights concerning the involvement of ER-stress and the accompanying UPR in health and in diseases. Problems to be addressed In this study, we seek to investigate if such a feedback loop may functionally exist, and if it would lead to amplification of ER-stress. These two problems are addressed by the following approaches. 1. To characterize the production of ROS in ER-stressed cells 2. To examine if oxidative stress developed during ER-stress would lead to amplification of ER-stress

Project Title:	The role of protein phosphatase on the regulation of messenger RNA translation in response to cellular stress
Investigator(s):	Wong NS
Department:	Biochemistry
Source(s) of Funding:	Seed Funding Programme for Basic Research
Start Date:	04/2009

Abstract:

Background: Metabolic insults arising from the extra-cellular environment often leads to perturbation of protein synthesis in the endoplasmic reticulum (ER). Consequently, nascent polypeptides are unable to undergo proper folding in the ER, forming instead misfolded protein species. The accumulation of these molecular species leads to impairment of ER metabolism, resulting in ER stress. Resolving ER stress is an important pre-requisite for a cell to finally adapt to metabolic insults and to continue to survive. The resolution of ER stress can be divided into two phases: a rapid but short lasting initial phase that is followed by a slow but longer lasting secondary phase. The initial phase is characterized by the rapid inhibition of messenger RNA translation, thereby reducing the protein folding load to the already stressed ER. Though an effective means of eliminating the source of ER stress, inhibition of mRNA translation can not last for a long time since many critical proteins essential for basic cell functions need to be synthesized continuously. Inhibition of mRNA translation is therefore only an interim measure but its implementation is critical to provide time for the longer lasting but slow-to-organize stress-relieving mechanisms (such as ER-associated degradation of proteins/ERAD and autophagy) to come into action. Key issues: The translation of mRNA is a complicated mechanism that can be divided into several stages. In the first stage, the small ribosomal subunit will bind to the 5’Cap of a protein-coding mRNA. This step is characterised by the formation of a ternary complex consisting of the eIF2, GTP and the met-tRNA. The formation of the eIF2-GTP-met-tRNA complex is one important site of regulation of mRNA translation. The eIF2 is composed of three non-identical subunits. One of those subunits, the α-subunit can undergo phosphorylation thereby abolishing the ability of eIF2 to assemble itself into the ternary complex. In the absence of such a complex, the small ribosomal subunit will not be able to bind to the mRNA to initiate translation. The phosphorylation of the α-subunit of eIF2 is the balance of the activities of specific protein kinases that act on eIF2α, as well as specific phosphatases that act on phospho-eIF2α. Although several protein kinases that are involved have been identified, the phosphatases that are involved are relatively uncharacterized. The translation of mRNA has to be regulated with high precision so that inhibition and resumption of this process will take place at the right moment within the stressed cell. Either a delay in the initiation, or the prolongation, of inhibition of mRNA translation could be detrimental to the stressed cell. Mechanisms underlying the regulation of mRNA translation are therefore an important issue for the understanding of how cells can successfully respond and adapt to ER stress. Problems being addressed: Several studies suggested that protein phosphatases involved in the regulation of phosphorylation of eIF2α are the serine/threonine phosphatases belonging to the PP1 family of protein phosphatases. This idea is consistent with the finding that eIF2α is phosphorylated at a serine residue (ser-51). However, at least three isoforms of PP1 are known and presently there is no data regarding which of the three isoforms is involved in the regulation of eIF2α phosphorylation. Secondly, PP1 and other protein serine/threonine phosphatase are previously shown to undergo inhibition by low-molecular-weight protein inhibitors, as well as inhibition via a redox mechanism. As ER stress is closely associated with the production of reactive oxygen species, the interesting question is raised as to whether the PP1 that is involved in the regulation of eIF2α phosphorylation is itself subject to regulation by ROS generated by ER stress. The objective of the present study is to examine the role of protein inhibitors and reactive oxygen species in the regulation of eIF2α phosphorylation.

List of Research Outputs

Lui W.T. and Wong N.S., Functional significance of the AU-rich sequence element in the mRNA of the stree-inducible protein CHOP/Gadd153, 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium, CUHK, Hong Kong, 15 May,. 2010.

Wong N.S., Chau M.C.H. and Chan L.K., Communication in medical education – harnessing the power of the Internet, Frontiers in Medical and Health Sciences Education,11-12 December 2009.

Researcher : Wong SYY

List of Research Outputs

Gao Y., Leung W.L., Wong S.Y.Y., Tam P.P.L., Chan D. and Cheah K.S.E., Type IIA Procollagen: A Positive Regulator of Nodal Signaling During Early Vertebrate Embryogenesis, 14th Research Postgraduate Symposium, December 2 & 3, 2009, The University of Hong Kong. 2009.

Researcher : Wong YME

List of Research Outputs

Wang X., Wong Y.M.E., Hui C.C. and Sham M.H., Hoxb1 cooperates with Gli to active Nkx2.2 expression in the neurogenesis of mammalian hind brain rhombomere 4., 43rd Annual Meeting for the Japanese Society of Developmental Biologists Kyoto, Japan, 20 June 2010 . 2010.

Researcher : Wu MH

List of Research Outputs

Cheung M.C.H., Chau K.H.B., Chan A.Y.L., Ng K.T., Wu M.H. and Briscoe J.A.M.E.S., Regulation of Sox9 Function by SUMOylation in avian neural crest development, In: Shinichi Aizawa Maria Leptin Nancy Papalopulu Claudio D Stern Dider Stainer Patrick Tam, Mechanisms of Development 16th International Society of Developmental Biologists Congress 2009 Book of Abstracts. Elsevier, 2009, 126: S308.

Kwok S.T.C., Wu M.H., Herve A., Nieto M.A. and Cheung M.C.H., The role of SoxE transcription factors in melanoma development, 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium, CUHK, Hong Kong, 15 May. 2010.

Wang Y., Lu L., Wu M.H., Sham M.H., Chan D., Cheah K.S.E. and Cheung M.C.H., In vitro generation of Osteo-chondroprogenitors, 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium, CUHK, Hong Kong, 15 May,. 2010.

Researcher : Wu X

List of Research Outputs

Wu X., Isthmin, a novel extracellular regulator in Nodal signaling pathway, PhD Thesis, 2010.

Researcher : Yalamanchili HK

List of Research Outputs

Wang J.J., Yang S., Yalamanchili H.K. and Li X., Co-evolution of transcription factor and its binding sites, Systems Biology: Global Regulation of Gene Expression, CSHL meeting, 23-27, March, 2010.

Yang S., Li X.R., Yalamanchili H.K. and Wang J.J., Co-evolution of transcription factors and their binding sites. 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium, Hong Kong. May 15, 2010.

Yang S., Li X.R., Yalamanchili H.K. and Wang J.J., Co-evolution of transcription factors and their binding sites, 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium, CUHK, Hong Kong, 15 May,. 2010.

Researcher : Yalamanchili HK

List of Research Outputs

Researcher : Yang L

List of Research Outputs

Tang H.C., Yang L., Tsang K.Y., Chan D. and Cheah K.S.E., Dual origin of osteoblasts in bone formation - terminally differentiated chondrocytes contribute to bone formation in vivo, 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium, CUHK, Hong Kong, 15 May,. 2010.

Researcher : Yang M

List of Research Outputs

Shi L., Yu B., Yang M. and Huang J., Engineering Tumor-killing Salmonella against Breast Cancer, 14th Research Postgraduate Symposium, December 2 & 3, 2009, The University of Hong Kong. 2009.

Researcher : Yang S

List of Research Outputs

Wang J.J., Yang S., Yalamanchili H.K. and Li X., Co-evolution of transcription factor and its binding sites, Systems Biology: Global Regulation of Gene Expression, CSHL meeting, 23-27, March, 2010.

Yang S., Li X.R., Yalamanchili H.K. and Wang J.J., Co-evolution of transcription factors and their binding sites. 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium, Hong Kong. May 15, 2010.

Yang S., Li X.R., Yalamanchili H.K. and Wang J.J., Co-evolution of transcription factors and their binding sites, 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium, CUHK, Hong Kong, 15 May,. 2010.

Yang S., Agrawal K.R., Lam T.W., Sham P.C., Cheah K.S.E. and Wang J.J., Novel Profile Hidden Markov Model to Predict MicroRNAs and their Targets Simutaneously, 14th Research Postgraduate Symposium, The University of Hong Kong, December 2-3. 2009.

Yang S., Agrawal K.R., Lam T.W., Sham P.C., Cheah K.S.E. and Wang J.J., Novel Profile Hidden Markov Model to predict microRNAs in Degenerative Disc Disease. University Grants Council site visit for Area of Excellence Programme "Developmental Genomics & Skeletal Research". January 25, 2010.

Researcher : Yang S

List of Research Outputs

Researcher : Yao KM

Project Title:	Regulation of Bmi-1 expression by the Forkhead box transcription factor FoxM1
Investigator(s):	Yao KM
Department:	Biochemistry
Source(s) of Funding:	Small Project Funding
Start Date:	11/2007

Abstract:

Mammalian cells enter a state of proliferative arrest known as senescence when subjected to various stresses including DNA damage, telomeric attrition, oxidative stress and overexpression of activated oncoproteins. Activation of the senescence program is associated with expression of b-galactosidase [referred as SA (senescence-associated)-b-gal activity] and the enlargement and flattening of cell morphology (reviewed in 1). Molecularly, activation of either or both of the p53-p21 and p16-Rb pathways are found to be necessary for the senescence response and senesence is believed to be a major mechanism behind cellular aging. Recently, various studies have also revealed senescence as an important tumor suppressor mechanism and the functional restoration of p53 as the master regulator of senescence in various tumor types is enough to promote tumor regression by triggering the senescence response (reviewed in 2). Further understanding the molecular basis of regulation of senescence will be of paramount importance to the design of strategies to suppress tumor formation and cellular aging. The Forkhead box (Fox) transcription factor FoxM1 is ubiquitously expressed in proliferating cells (3, 4, 5). FoxM1 expression is initiated just before entry into S phase but its levels peak at the G2/M phase of the cell cycle. FoxM1 deficiency in mice and cultured cells leads to defects in mitosis and FoxM1 directly up-regulates the expression of target genes like Cyclin B1, Cdc25B, Plk1 and Aurora B, which are essential for driving cells into mitosis (reviewed 6). It is interesting to note that early-passage mouse embryonic fibroblasts (MEFs) derived from homozygous FoxM1 knockout mice underwent premature senescence with expression of high levels of SA-b-gal and p19ARF (7). This information suggests involvement of FoxM1 in the regulation of cellular senescence but the molecular mechanism underlying the onset of senescence in FoxM1-deficient MEFs remains unclear. To investigate the regulatory role of FoxM1 in stress-induced premature senescence (SIPS), we have recently established two doxycycline (dox)-inducible FoxM1-expressing mouse embryonic fibroblastic (NIH3T3) cell lines. Treatment of these lines with sub-lethal doses (20 uM and 100 uM) of H2O2 induced senescence with SA-b-gal expression and elevated levels of p53 and p21. Interestingly, induction of FoxM1 expression dramatically suppressed SIPS and expression of p53 and p21. Consistent with a down-regulation of the p53-p21 pathway, expression of p19ARF, which is a well-known positive regulator of p53 stability, is decreased while levels of the Polycomb group protein Bmi-1 were induced. Bmi-1 is a major negative regulator of the Ink4a/Arf/Ink4b locus which encodes expression of p19ARF as well as the cyclin-dependent kinase inhibitors p16INK4a and p15INK4b. This analysis indicated that increased FoxM1 expression in MEFs exerted an antagonistic effect against oxidative SIPS by suppressing the p53-p21 pathway. This finding revealed an active role played by FoxM1 in protecting against senescence by suppressing p53 and p19AF expression via induction of Bmi-1 levels. In this application, I propose to conduct experiments to investigate the regulation of Bmi-1 expression by FoxM1. First, regulation of Bmi-1 by FoxM1 will be verified by immunoblot and semi-quantitative RT-PCR analysis using the dox-inducible cell lines. Second, FoxM1 in NIH3T3 cells will be depleted using shRNAs to investigate whether Bmi-1 is a physiological target of FoxM1. Third, levels of FoxM1 and Bmi-1 at different cell cycle phases will be correlated to substantiate the regulatory effect of FoxM1 on Bmi-1. Fourth, transient reporter assays will be conducted to test the activating effect of FoxM1 on the Bmi-1 promoter. Taken together, the proposed experiments will reveal whether Bmi-1 is a direct regulatory target of FoxM1 and will provide better understanding of the anti-senescence effect of FoxM1, which are of major biological interest and of relevance to the future development of strategies to suppress tumor formation and cellular aging. References: 1. Zhang H (2007) J Cell Physiol 210:567-574. 2. Dimri G (2005) Cancer Cell 7: 505-512. 3. Korver W, Roose J, and Clevers H (1997) Nucleic Acids Res 25: 1715-1719. 4. Ye H, Kelly TF, Samadani U, Lim L, Rubio S, Overdier DG, Roebuck KA, and Costa RH (1997) Mol Cell Biol 17: 1626-1641. 5. Yao KM, Sha M, Lu Z, and Wong GG (1997) J Biol Chem 272: 19827-19836. 6. Laoukili J, Stahl M, and Medema RH (2007) Biochim Biophy Acta 1775: 92-102. 7. Wang IC, Chen YJ, Hughes D, Petrovic V, Major ML, Park HJ, Tan Y, Ackerson T, and Costa RH (2005) Mol Cell Biol 25: 10875-10894.

Project Title:	Characterization of the interaction between Pdzd2 and Kir6.2
Investigator(s):	Yao KM
Department:	Biochemistry
Source(s) of Funding:	Small Project Funding
Start Date:	09/2008
Completion Date:	02/2010

Abstract:

Insulin produced by pancreatic β cells is the key hormone responsible for maintaining glucose homeostasis. Upon the challenge of a stimulus, e.g. glucose, exocytosis of insulin is triggered. Such glucose-induced insulin secretion is commonly distinguished as a biphasic process which depends on the rise of [Ca2+]i. As the extracellular glucose concentration increases above 6 mM, glucose enters the cells through Glut2 transporters. Subsequent degradation of glucose causes an elevation in the ATP/ADP ratio, and hence directs the closure of the KATP channel. Followed by membrane depolarization, the influx of Ca2+ activates the exocytosis of insulin granules. As defective or deficient insulin secretory responses that result from impaired pancreatic β-cell functions lead to diabetes mellitus (1, 2), an understanding of the regulatory mechanisms of glucose-stimulated insulin secretion (GSIS) is definitely relevant to the pathogenesis and treatment of diabetes. Pdzd2 [previously named KIAA0300 (3), PIN-1 (4), PAPIN (5), activated in prostate cancer (AIPC, 6)] is a multi-PDZ protein expressed in multiple tissues including heart, brain, lung and pancreas (7). Recent immunohistochemical analyses revealed selective expression of Pdzd2 in pancreatic islet β cells, but not in exocrine or glucagon-secreting α cell (8). Pdzd2 cDNAs were originally cloned based on the protein’s binding and expression properties but its function remains obscure as no functional analysis in animal or cell model has been reported. Pdzd2 contains six different PDZ protein-protein interaction domains, which are thought to function by acting as molecular scaffolds to facilitate the assembly of macromolecular complexes. Pdzd2 shows the strongest sequence match to pro-interleukin-16 at the carboxy-terminus (7) but is most structurally similar to the longer neuronal form of pro-interleukin16 (npro-IL-16, 9), which is a 5-PDZ-domain protein. Npro-IL-16 has been reported to interact with NMDA receptor subunit 2A and Kv4 channels in interaction assays (9). The interactions of npro-IL-16 with Kv4.2 in the hippocampus and cerebellum were recently found to induce intracellular clustering of Kv4.2 and hence modulate its channel function (10). The structural similarity between npro-IL-16 and Pdzd2 implies a potential intracellular role for Pdzd2, acting as a scaffolding protein for targeting receptors or ion-channels. To explore the in vivo function of Pdzd2, Pdzd2-deficient mice were recently generated from two lines of ES cells with the Pdzd2 locus disrupted by retroviral insertion. Homozygous Pdzd2 mutant mice were viable and appeared grossly morphologically normal. When analyzed for glucose homeostasis, Pdzd2-deficient mice showed enhanced glucose tolerance in intraperitoneal glucose tolerance tests. Their plasma insulin levels indicated increased insulin secretion after fasting and during the first phase of GSIS. The abnormality in insulin secretion was further investigated in ex-vivo assays using isolated pancreatic islets. Insulin release from mutant islets measured at 2.8 mM of glucose was found to be two-fold higher than normal islets. To verify the functional defect in vitro, Pdzd2 was depleted in INS-1E cells using two siRNA duplexes. Pdzd2-depleted INS-1E cells also displayed increased insulin secretion at low concentrations of glucose. Importantly, the Pdzd2-dependent defect was lost when KATP channel closure was promoted by tolbutamine treatment and co-immunoprecipitation analysis showed that Pdzd2 and the KATP channel subunit Kir6.2 interact in vivo. Taken together, our results provide the first evidence that Pdzd2 is required for normal regulation of GSIS and that this regulatory effect is exercised via modulation of KATP channel activity. We hypothesize that Pdzd2 modulates KATP channel function by directly interacting with Kir6.2 and affecting its surface expression. This proposal aims to further investigate the interaction between Pdzd2 and Kir6.2 and how this interaction may affect Kir6.2 function. First, cDNAs encoding epitope-tagged Pdzd2 and Kir6.2 will be transfected into COS-7 cells to test for direct interaction by immunoprecipitation using anti-epitope antibodies. Second, deletion and substitutive mutants of Pdzd2 and Kir6.2 will be constructed to identify the interaction domains within the two proteins. Third, wildtype and mutant epitope-tagged Pdzd2 and Kir6.2 cDNAs will be transfected singly or in combination into COS-7 cells to explore the ability of Pdzd2 to affect the surface expression of Kir6.2. Fourth, Kir6.2 surface expression will be studied in Pdzd2-depleted INS-1E cells to explore the in vivo relevance of the Pdzd2 regulatory effect detected in vitro. The proposed experiments will reveal whether Pdzd2 interacts directly with Kir6.2 and modulates its expression in insulin-secreting beta-like cells, which is of major biological interest and of relevance to the future development of strategies to treat diabetes. References: 1. Ashcroft FM 2005 ATP-sensitive potassium channelopathies: focus on insulin secretion. J Clin Invest 115: 2047-2058 2. Koster JC, Permutt MA, Nichols CG 2005 Diabetes and insulin secretion: the ATP-sensitive K+ channel (K ATP) connection. Diabetes 54: 3065-3072 3. Nagase T, Ishikawa K, Nakajima D, Ohira M, Seki N, Miyajima N, Tanaka A, Kotani H, Nomura N, Ohara O 1997 Prediction of the coding sequences of unidentified human genes. VII. The complete sequences of 100 new cDNA clones from brain which can code for large protein in vitro. DNA Res 4: 141-150 4. Thomas MK, Yao KM, Tenser MS, Wong GG, Habener JF 1999 Bridge-1, a novel PDZ-domain interactor of E2A-mediated regulation of insulin gene transcription. Mol Cell Biol 19: 8492-8504 5. Deguchi M, Iizuka T, Hata Y, Nishimura W, Hirao K, Yao I, Kawabe H, Takai Y 2000 PAPIN. A novel multiple PSD-95/Dlg-A/ZO-1 protein interacting with neural plakophilin-related armadillo repeat protein/δ-catenin and p0071. J Biol Chem 275: 29875-29880 6. Chaib H, Rubin MA, Mucci NR, Li L, Taylor JMG, Day ML, Rhim JS, Macoska JA 2001 Activated in prostate cancer: a PDZ domain-containing protein highly expressed in human primary prostate tumors. Cancer Res 61: 2390-2394 7. Yeung ML, Tam TS, Tsang AC, Yao KM 2003 Proteolytic cleavage of PDZD2 generates a secreted peptide containing two PDZ domains. EMBO Rep 4: 412-418 8. Ma RY, Tam TS, Suen AP, Yeung PM, Tsang SW, Chung SK, Thomas MK, Leung PS, Yao KM 2006 Secreted PDZD2 exerts concentration-dependent effects on the proliferation of INS-1E cells. Int J Biochem Cell Biol 38: 1015-1022 9. Kurschner C, Yuzaki M 1999 Neuronal interleukin-16 (NIL-16): a dual function PDZ domain protein. J Neurosci 19: 7770-7780 10. Fenster CP, Fenster SD, Leahy HP, Kurschner C, Blundon JA 2007 Modulation of Kv4.2 K+ currents by neuronal interleukin-16, a PDZ domain-containing protein expressed in the hippocampus and cerebellum. Brain Res 1162: 19-31

Project Title:	Test of FoxM1 as an enhancer of induced pleuripotent stem cell production
Investigator(s):	Yao KM, Cheung MCH
Department:	Biochemistry
Source(s) of Funding:	Seed Funding Programme for Basic Research
Start Date:	03/2010

Abstract:

The recent establishment of protocols for reprogramming of somatic cells to pleuripotent stem cells has ushed in a new era of cell biology. First reported by Dr. Shinya Yamanaka at Kyoto University, the introduction of four transcription factors (Oct4, Sox2, c-Myc and Klf4) into mouse embryonic fibroblasts (MEFs) was enough to direct formation of induced pluripotent stem (iPS) cells with properties characteristics of embryonic stem cells (reviewed in Yamanaka, 2007). Since the initial discovery in 2007, extensive efforts have been made to improve the reprogramming efficiency and to apply the technology to human cells with the ultimate goal of generating patient-specific stem cells for the analysis and treatment of diseases. Recently, it was discovered that iPS reprogramming efficiency could be improved by inactivation of the p53 tumor suppressor pathway (Hong et al., 2009; Li et al., 2009; Kawamura et al., 2009; Utikal et al., 2009, Marion et al., 2009) and ablation of other related senescence effectors like p16INK4a and p21CIP1 (Banito et al., 2009). Unfortunately, these p53- or senescence-deficient iPS cells were genomically unstable and could not produce progeny mice efficiently, an outcome which is not surprising based on the known protective functions of p53 and various senescence pathways. Therefore, there is an urgent need to design strategy to increase the efficiency of iPS reprogramming without causing genome instability. The Forkhead box M1 (FoxM1) transcription factor is known to be proliferation-associated (Wierstra and Alves, 2007). Loss-of-function analyses in FoxM1 knockout mice and various FoxM1-depleted cells have demonstrated an essential role of FoxM1 in promoting cell proliferation at both the S and M phases of the cell cycle. As perturbation of FoxM1 function in various cancer models slows down tumorigenesis, FoxM1 has been regarded as an oncoprotein extensively investigated as a target for cancer therapy. However, recent studies have revealed a more positive side of FoxM1 function. FoxM1 was found to be a target of Chk2 after activation of the DNA damage response, and Chk2 phosphorylation of FoxM1 leads to its stabilization and upregulation of the DNA repair genes XRCC1 (X-Ray Cross-Complementing group 1) and BRCA2 (Breast Cancer-Associated gene 2)(Tan et al., 2007). Interestingly, FoxM1 depletion was shown to result in increased DNA damage, p53 activation and premature cellular senescence (Tan et al., 2007) and FoxM1 overexpression was found to counteract oxidative stress-induced senescence by downregulation of the p19Arf-p53 pathway (Li et al., 2008). Most recently, FoxM1 expression was shown to be negatively regulated by p53 (Barsotti and Prives, 2009; Pandit et al., 2009) further supporting the opposing effects of FoxM1 and p53 against the action of each other. As suppression of p53 function and/or senescence pathways was found to increase the efficiency of iPS reprogramming, we wonder whether FoxM1 overexpression can be used as a strategy to enhance reprogramming of MEFs into pleuripotent stem cells. We hypothesize that FoxM1, which has a counteracting effect against p53 and senescence, can enhance generation of iPS cells when coexpressed with the Yamanaka factors. In this study, we propose infection of MEFs using retroviruses carrying FoxM1 and the Yamanaka factors. MEFs will be tagged with an Oct4-mCherry red reporter construct and the efficiency of iPS cell formation will be assessed by counting alkaline-phosphatase-positive and Oct4-positive colonies. Expression of various reprogramming markers will also be studied by RT-PCR and immunocytochemical analyses to monitor the progress of iPS reprogramming. To study whether FoxM1 coexpression has any effect on DNA damage and p53 expression, formation of DNA breaks will be assayed by immunostaining for γH2AX, 53BP1, and TUNEL foci, and p53, using commercially available antibodies. These analyses will reveal whether FoxM1 can function as a reprogramming-promoting factor while avoiding the drawback of genome instability usually associated with p53 suppression. References Banito A et al. (2009) Genes & Dev 23: 2134-2139. Barsotti AM and Prives C (2009) Oncogene, in press. Hong H et al. (2009) Nature 460: 1132-1135. Kawamura T et al. (2009) Nature 460: 1140-1144. Li H et al. (2009) Nature 460: 1136-1139. Li SKM et al. (2008) J Biol Chem 283: 16545-16553. Marion RM et al. (2009) Nature 460: 1149-1153. Pandit B, Halasi M and Gartel AL (2009) Cell Cycle 8: 3425-3427. Tan Y, Raychaudhuri P and Costa RH (2007) Mol Cell Biol 27: 1007-1016. Utikal J et al. (2009) Nature 460: 1145-1148. Wierstra I and Alves J (2007) Biol Chem 388: 1257-1274. Yamanaka S (2007) Cell Stem Cell 1: 39-49.

Project Title:	43rd Annual Meeting for the Japanese Society of Developmental Biologists Secreted PDZ Domain-containing Protein 2 (sPDZD2) exerts insulinotropic effects on INS-1E cells via a protein kinase A-dependent mechanism
Investigator(s):	Yao KM
Department:	Biochemistry
Source(s) of Funding:	URC/CRCG - Conference Grants for Teaching Staff
Start Date:	06/2010
Completion Date:	06/2010

Abstract:

N/A

List of Research Outputs

Chan C.Y., Tsang S.W. and Yao K.M., Secreted PDZ Domain-containing Protein 2 (sPDZD2) Exerts Insulinotropic Effects on INS-1E Cells via a Protein Kinase A-Dependent Mechanism, Japan Society of Developmental Biology 2010 at Kyoto, Japan. 2010.

Chan D.W., Liu V.W.S., To M.Y., Chiu P.M., Lee Y.W., Yao K.M., Cheung A.N.Y. and Ngan H.Y.S., Overexpression of FOXG1 contributes to TGF-beta resistance through inhibition of p21(WAF1/CIP1) expression in ovarian cancer, Briitish Journal of Cancer. 2009, 101: 1433-1443.

Cui J., Wang Z., Cheng Q.N., Lin R., Zhang X., Leung P.S., Copeland N.G., Jenkins N.A., Yao K.M. and Huang J., Targeted Inactivation of KIF5B in Pancreatic Leads to Insulin Secretory Deficiency and Increased b-Cells Mass in vivo, 14th Research Postgraduate Symposium, December 2 & 3, 2009, The University of Hong Kong. 2009.

Lam K.Y. and Yao K.M., Differential Regulation of FoxM1 Isoforms by Raf/MEK/ERK Signaling, 14th Research Postgraduate Symposium, December 2 & 3, 2009, The University of Hong Kong. 2009.

Shao D., Baker M.D., Abrahamsen B., Rugiero F., Malik-Hall M., Poon W.Y., Cheah K.S.E., Yao K.M., Wood J.N. and Okuse K., A multi PDZ-domain protein Pdzd2 contributes to functional expression of sensory neuron-specific sodium channel Nav1.8, Molecular and Cellular Neuroscience. Elsevier, 2009, 42: 219-225.

Shiu S.Y.W., Pang B., Tam C.W. and Yao K.M., Involvement of Gas and Gaq proteins in melatonin-induced prostate epithelial cell antiproliferation, XI. Congress of the European Biological Rhythms Society, Strasbourg, France, August 22-28. 2009, S10-5.

Tsang S.W., Shao D., Cheah K.S.E., Okuse K., Leung P.S. and Yao K.M., Increased basal insulin secretion in Pdzd2-deficient mice, Mol Cell Endocrinol. 2010, 315(1-2): 263-70.

Researcher : Yeung MN

List of Research Outputs

Yeung M.N., Functional roles of heparanase in endochondral ossification, PhD Thesis. 2009.

Researcher : Yu B

List of Research Outputs

Shi L., Yu B., Yang M. and Huang J., Engineering Tumor-killing Salmonella against Breast Cancer, 14th Research Postgraduate Symposium, December 2 & 3, 2009, The University of Hong Kong. 2009.

Researcher : Zhang M

List of Research Outputs

Chu K.H., Zhang M., Wong E.Y.M., Szeto Y.Y., Chan Y.S., Cheah K.S.E. and Sham M.H., Best Poster Award: Sox10 mutation affects cochleo-vestibular ganglion gliogenesis, 43rd Annual Meeting for the Japanese Society of Developmental Biologists, Kyoto, Japan, 20-23 June, 2010.. 2010.

Chu K.H., Zhang M., Wong E.Y.M., Szeto Y.Y., Chan Y.S., Cheah K.S.E. and Sham M.H., Best Poster Award: Sox10 mutation affects gliogenesis of the cochleo-vestibular ganglion, 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium, 15 May 2010. 2010.

Chu K.H., Zhang M., Wong E.Y.M., Szeto Y.Y., Chan Y.S., Cheah K.S.E. and Sham M.H., Sox10 mutation affects cochleo-vestibular ganglion gliogenesis, 43rd Annual Meeting for the Japanese Society of Developmental Biologists, Kyoto, Japan, 20-23 June. 2010.

Chu K.H., Zhang M., Wong E.Y.M., Szeto Y.Y., Chan Y.S., Cheah K.S.E. and Sham M.H., Sox10 mutation affects gliogenesis of the cochleo-vestibular ganglion, 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium, 15 May 2010.

Zhang M., Lui V.C.H., Tam P.K.H. and Sham M.H., Abnormal Enteric Nervous System Development in a Sox10^NGFP Mouse Mutant, 14th Research Postgraduate Symposium, December 2 & 3, 2009, The University of Hong Kong. 2009.

Zhang M., Leung C., Lui V.C.H., Tam P.K.H. and Sham M.H., Sox10 affects enteric neural crest cells migration in a Sox10NGFP/+ mutant , 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium, CUHK, Hong Kong, 15 May,. 2010.

Zhang M., Leung C., Lui V.C.H., Tam P.K.H. and Sham M.H., Sox10 is required for proliferation and migration of enteric neural crest stem cells, International Society for Stem Cell Research, 8th Annual Meeting, Moscone West, San Francisco, CA, USA, June 16-19, 2010.

Zhang M., Leung C., Lui V.C.H., Tam P.K.H. and Sham M.H., Sox10 mediates proliferation and migration behavior of enteric neural crest cells, 43rd Annual Meeting for the Japanese Society of Developmental Biologists, Jointly Sponsored by the Asia-Pacific Developmental Biology Network, Kyoto, Japan, 20-23 June. 2010.

Researcher : Zhang M

List of Research Outputs

Researcher : Zhang X

List of Research Outputs

Cui J., Wang Z., Cheng Q.N., Lin R., Zhang X., Leung P.S., Copeland N.G., Jenkins N.A., Yao K.M. and Huang J., Targeted Inactivation of KIF5B in Pancreatic Leads to Insulin Secretory Deficiency and Increased b-Cells Mass in vivo, 14th Research Postgraduate Symposium, December 2 & 3, 2009, The University of Hong Kong. 2009.

Researcher : Zhang Y

List of Research Outputs

Cham W.C., Ma C.W., Zhang Y., Xie H., Yung W.H., Chan Y.S. and Shum D.K.Y., The Regulatory Role of Heparanase in Synaptic Plasiticity at Hippocampus, 14th Research Postgraduate Symposium, December 2 & 3, 2009, The University of Hong Kong. 2009.

Cham W.C., Ma C.W., Zhang Y., Xie H., Yung W.H., Chan Y.S. and Shum D.K.Y., The regulatory role of heparanase of synaptic plasticity in hippocampus, Society Neuroscience Abstract (USA). 2009, 318.3/C4.

Ma C.W., Zhang Y., Cham W.C., Chan Y.S. and Shum D.K.Y., Role of heparanase in synaptic plasticity at the hippocampus and vestibular nucleus , J. Physiological Sciences (Suppl 1). [36^th Int’l Congress of International Union of Physiological Sciences Kyoto, Japan. July 27 - Aug. 1, 2009]. 2009, 59: 145.

Zhang Y., Zhang Y. and Song Y., The role of Pax6 in Beta-Amyloid-induced neuronal apoptosis, 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium, CUHK, Hong Kong, 15 May,. 2010.

Researcher : Zhang Y

List of Research Outputs

Researcher : Zhang Y

List of Research Outputs

Researcher : Zhang Y

List of Research Outputs

Researcher : Zhou J

List of Research Outputs

Guo S., Zhou J., Gao B., Hu J., Wang H., Meng J., Zhao X., Ma G., Lin C., Xiao Y., Tang W., Zhu X., Cheah K.S.E., Feng G., Chan D. and He L., Missense mutations in IHH impair Indian Hedgehog signaling in C3H10T1/2 cells: Implications for brachydactyly type A1, and new targets for Hedgehog signaling, Cell Mol Biol Lett. 2009, 15(1): 153-76.

Zhou J. and Zhou Z., iPS Reprogramming in Premature Aging Mouse Model-Zmpste24 Deficient Mice, 14th Research Postgraduate Symposium, December 2 & 3, 2009, The University of Hong Kong. 2009.

Researcher : Zhou J

List of Research Outputs

Zhou J. and Zhou Z., iPS Reprogramming in Premature Aging Mouse Model-Zmpste24 Deficient Mice, 14th Research Postgraduate Symposium, December 2 & 3, 2009, The University of Hong Kong. 2009.

Researcher : Zhou S

List of Research Outputs

Zhou S., Defects in Early B Lymphocyte Development in Zmpste24^-/-Mice, MPhil Thesis. 2009.

Researcher : Zhou Z

Project Title:	Understanding the regulation of MT1-MMP activity in tumorigenesis
Investigator(s):	Zhou Z
Department:	Biochemistry
Source(s) of Funding:	Matching Fund for NSFC Young Researcher Award
Start Date:	05/2006

Abstract:

To investigate the effect of mis-regulated MT1-MMP activity on development and tumorigenesis; to understand how the mis-regulated MT1-MMP activity regulate FGF signaling which contributes to the defective development and disturbed angiogenesis.

Project Title:	Triggering senescence in tumor cells via unprocessed lamin A
Investigator(s):	Zhou Z, Wong WT, Kao RYT
Department:	Biochemistry
Source(s) of Funding:	General Research Fund (GRF)
Start Date:	09/2006
Completion Date:	08/2009

Abstract:

To investigate whether FTI treatment causes senescence, genomic instability and/or DNA repair defects in tumor cells; to assess the contribution of unprocessed prelamin A to the inhibitory effect of FTIs on tumor growth; to examine whether FTIs can directly inhibit Zmpste24 activity.

Project Title:	Lamin C in development and tumorigenesis
Investigator(s):	Zhou Z, Chan LC
Department:	Biochemistry
Source(s) of Funding:	General Research Fund (GRF)
Start Date:	09/2007

Abstract:

To generate lamin C conditional knockout mice ; to analyze phenotypes of these lamin C conditional knockout mice to investigate role for lamin C in development, lymphocyte differentiation and transformation; to examine effects of lamin C deficiency on DNA repair; to investigate the potential links between lymphocyte tumorigenesis and altered epigenetics/ stem cell/progenitor self-renewal in lamin C knockout mice.

Project Title:	Screening anti-ageing substances in small chemical library using premature ageing animals
Investigator(s):	Zhou Z
Department:	Biochemistry
Source(s) of Funding:	Innovation and Technology Fund Internship Programme
Start Date:	09/2007

Abstract:

1) Establish a high-throughput assay system to screen substances that can prevent the earlier sescence in Zmpste24-/- cells. 2) Identify potentially safe substances and further test their anti-ageing in premature ageing mice. 3) To investigate the molecular mechanism of anti-ageing effect of the most effective substance found.

Project Title:	Aging: epigenetic regulation, cell cycle checkpoint control and stem cell potency
Investigator(s):	Zhou Z, Cheah KSE, Chu IK, Smith DK, Lui VCH
Department:	Biochemistry
Source(s) of Funding:	Collaborative Research Fund (CRF) - Group Research Project
Start Date:	03/2008

Abstract:

1) To uncover specific histone modifications that are critical for both natural aging and premature aging. i) histone modifications in aging and premature aging; ii) test specific histone modification on DNA repair and aging. 2) To determine histone modifications that are the “driving force of aging” and their potential key targeting genes or pathways and to test in a model system. 3) To determine if accumulation of unprocessed/truncated forms of prelamin A affects the checkpoints that monitor proper DNA replication and mitosis. i) spindle-assembly checkpoint in laminopathy-based premature aging; ii) DNA replication checkpoint and prelamin A-based premature aging. 4) To investigate defective stem cell self-renewal and the contribution of stem cell decline to aging. i) to determine whether stem/progenitor cells decline in premature aging; ii) to determine whether stem cell transplantation ameliorates premature aging phenotypes and prolongs the lifespan in premature aging mice; iii) to determine mechanism of stem cells decline in premature aging.

Project Title:	Regulation of FGFR signaling by MT1-MMP
Investigator(s):	Zhou Z
Department:	Biochemistry
Source(s) of Funding:	General Research Fund (GRF)
Start Date:	09/2008

Abstract:

(1) To determine the specific cleavage site of FGFR2 in MT1-MMP deficient COs in order to find out the responsible protease; (2) To determine the contribution of FGFR2 truncation to defective calvaria bone development in MT1-MMP deficient embryos and how MT1-MMP contribute to the FGFR2 truncation. (In this part, we will NOT generate BAC transgenic mice that expressing truncated FGFR2 (Objective 2.4); (3) To directly test if Adam9 is involved in this specific proteolytic processing of FGFR2. (In this part, we will NOT make MT1-MMP deletion construct to test the binding and protection effects of MT1-MMP toward FGFR.

Project Title:	Gordon Research Conference on Matrix Metalloproteinase Membrane Type 1-Matrix Metalloproteinase regulates FGFR signaling through inactivation of ADAM9 in osteogenesis
Investigator(s):	Zhou Z
Department:	Biochemistry
Source(s) of Funding:	URC/CRCG - Conference Grants for Teaching Staff
Start Date:	08/2009

Abstract:

N/A

List of Research Outputs

Azar D.T., Casanova F.H., Mimura T., Jain S., Zhou Z., Han K.Y. and Chang J.H., Corneal epithelial MT1-MMP inhibits vascular endothelial cell proliferation and migration., Cornea. . 2010, 29(3): 321-30.

Gawden-Bone C., Zhou Z., King E., Prescott A., Watts C. and Lucocq J., Dendritic cell podosomes are protrusive and invade the extracellular matrix using metalloproteinase MMP-14., J Cell Sci.. 2010, 123(9): 1427-37.

Zhou J. and Zhou Z., iPS Reprogramming in Premature Aging Mouse Model-Zmpste24 Deficient Mice, 14th Research Postgraduate Symposium, December 2 & 3, 2009, The University of Hong Kong. 2009.

Zhou Z., Academic Editor, PLoS ONE. 2010.

Zhou Z., Epigenetic regulation of premature aging, Special Workshop on Cutting Edge Science in Aging Research. SEOUL, KOREA May 24, 2010. 2010.

Zhou Z., Impaired Chromatin Modification Interferes With Double-Strand Break Repair In Accelerated Aging Syndrome, Frontiers Biomedical Research, HKU. December 4. 2009.

Zhou Z., Impaired Chromatin Remodeling Interferes With Dna Double Strand Break Repair In Premature Aging Syndrome, Cell Cycle Regulation And Tumorigenesis Symposium/imcb, Singapore. Singapore, 2009.

Zhou Z., Jin G. and Chan J.K.M., Mt1-mmp Regulates Fgf Signaling In Osteogenesis, Gordon Research Conference-Matelloproteinses. Les Diablerets, Switzerland, 2009.

-- End of Listing --