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Infect Control Hosp Epidemiol 12, 96-100. 190 Appendix A Common buffers, solutions and media All solutions were dissolved in ddH2O and were autoclaved or filter sterilized unless otherwise specified. 1% Agarose gel 1% (w/v) Agarose 0.5xTAE Buffer 0.25µg/ml Ethidium bromide 2% Agarose gel 2% (w/v) Agarose 0.5xTAE Buffer 0.25µg/ml Ethidium bromide BCIG/IPTG plates 20 µl BCIG (20 mg/ml) 20 µl IPTG (20 mg/ml) spread plate immediately Carbonate coating buffer per liter, 0.05 M 5.3 g anhydrous Na2CO3 adjust to pH9.6 10XCIP dephosphorylation buffer (Boehringer Mannheim) 0.5M Tris.Cl 1mM Ethylenediaminetetraacetic acid (EDTA) pH8.5 (at 20oC) Denaturing solution 1.5M Sodium chloride 0.5M Sodium hydroxide 100XDenhardt’s solution 2% (w/v) Bovine serum albumin (BSA) 2% (w/v) Ficoll 2% (w/v) Polyvinylpyrrolidone 10XEE 100mM EPPS 10mM EDTA, pH8.0 EEN 2.5xEE 0.5M NaCl LB (Luria-Bertani) media 1% (w/v) Tryptone 0.5% (w/v) Yeast extract 191 0.5% (w/v) Sodium chloride 1.5% (w/v) Bacto-agar (plates only) LB/Ampicillin LB 100µg/ml Ampicillin 1.5% (w/v) Bacto-agar (plates only) LB/Glycerol (for -70oC storage of bacterial stocks) 50% (v/v) LB 50% (v/v) Glycerol LB/Kanamycin LB 50µg/ml Kanamycin 1.5% (w/v) Bacto-agar (plates only) Loading dye per ml in 1X TE buffer 0.25 % (w/v) Bromophenol Blue, 7.5 mg 20% Glycerol 0.1mg/ml RNase A (DNase-free) Lysis solution 5% Sodium dodecyl sulphate (SDS) 50mM Tris, pH8.0 62.5mM EDTA, pH8.0 Minimal media (MM) 1.5% (w/v) Bacto-agar, autoclaved 1xMinimal Salts 0.5% (w/v) Glucose 0.1% (w/v) Magnesium chloride 10XMinimal Salts 7.0% (w/v) K2HPO4 3.0% (w/v) KH2PO4 1.0% (w/v) (NH4)2SO4 0.5% (w/v) Sodium citrate.3H2O pH7.5 (adjusted with sodium hydroxide) Neutralising solution 1.5M Sodium chloride 0.5M Tris.Cl, pH7.2 1mM EDTA 10XPBS 8% (w/v) Sodium chloride, g/L 0.2% (w/v) Potassium chloride, 0.2 g/L 1.44% (w/v) Na2HPO4, 1.44 g/L 0.24% (w/v) KH2PO4, 0.24 g/L Adjust to pH7.4 192 Pre-hybridisation solution 5xSSPE 5xDenhardt’s solution 0.5% (w/v) Sodium dodecyl sulphate (SDS) Protein purification lysis, wash and elution buffers derived from the QIAexpressionist handbook (Qiagen) SDS-PAGE destaining solution per liter 100 ml methanol 100 ml glacial acetic acid 800 ml ddH2O SDS-PAGE loading buffer 50 mM Tris-HCl, pH8.0 2% SDS 0.1% bromophenol blue 10% glycerol 4% mercaptoethanol, final concentration SDS-PAGE staining solution per liter 2.5 g Coomassie brilliant blue R250 360 ml methanol 80 ml glacial acetic acid 360 ml ddH2O Solution I 50mM Glucose 25mM Tris-HCl, pH8.0 10mM EDTA, pH8.0 Solution II 0.2M Sodium hydroxide 1% Sodium dodecyl sulphate (SDS) freshly prepared from 10N NaOH and 10% SDS stocks Solution III per 100 ml, pH4.8 60% (v/v) 5M Potassium Acetate, 60 ml 11.5% (v/v) Glacial acetic acid, 11.5 ml ddH2O, 28.5 ml 20XSSC 3M Sodium chloride 0.3M Sodium citrate.3H2O 20XSSPE 3.6M Sodium chloride 0.2M Sodium phosphate 0.02M EDTA, pH7.7 193 10XT4 DNA ligase buffer 250mM Tris.Cl 100mM Magnesium chloride 2mM Dithiothreitol (DTT) 4mM ATP pH7.8 (at 37oC) 10XTaq DNA Polymerase buffer 100mM Tris.Cl 15mM Magnesium chloride 500mM Potassium chloride pH9.0 50X TAE buffer per liter 242 g Tris, pH8.0 57.1 ml glacial acetic acid 0.05M EDTA, pH8.0 TBS per liter, pH7.4 g NaCl 0.2 g KCl 3.0 g TES buffer 50mM Tris, pH8.0 5mM EDTA, pH9.0 50mM Sodium chloride 194 Appendix B Curriculum Vitaé Profession : Molecular Microbiologist/Medical Lab. Microbiologist PERSONAL DATA Name Date of Birth Present Address : : : E-mail Address : EDUCATION Post-Graduate DARIO CRUZ ANGELES December 16, 1965 Microbiology Department, Faculty of Medicine The National University of Singapore Science Drive 2, 117597, Singapore Tel. (65) 9049-9938 Fax. (65) 6776-6872 medp0194@nus.edu.sg : Master of Philosophy in Biochemistry and Molecular Biology The Australian National University School of Biochemistry and Molecular Biology Faculty of Science, Canberra, 0200 Australian Capital Territory January 1999 – March 2001 (AUSAID Scholar) : Master of Science in Public Health (Medical Microbiology) University of the Philippines, College of Public Health Department of Medical Microbiology, Phil. General Hospital Pedro Gil Street, Ermita, Manila June 1987- April 1992 (UP-GIA Scholar) : Advance Genetics and Cell Biology Course, University of the Philippines, Institute of Biology March-May 1998 (UP-GIA Scholar) Tertiary : Bachelor of Science in Biology University of the Philippines, Institute of Biology June 1983- March 1987 (College Scholar, Dean’s List) Secondary : MIT Pre-Engineering High School, 1979-1983 (Valedictorian, Editor-in-Chief) Primary : Northern Yorklin Chinese School, 1971-1979 (Valedictorian, Gold Medalist) ELIGIBILITY Technologist in Microbiology : Microbiology (Passed, #7220981) American Society for Clinical Pathology Board of Registry P.O. Box 12277 Chicago IL 60612-0277 195 : Registered Microbiologist (Passed, #5285176) Clinical and Public Health Category American Academy for Microbiology National Registry of Microbiologists (ASM) 1325 Massachusetts Avenue, N.W. Washington D.C., 20005-4171 USA RegisteredMedical Technologist: Medical Technologist (Passed, #83078) American Medical Technologists 710 Higgins Road, Park Ridge Illinois, 60068-5765 USA : Membership American Society for Microbiology Philippine Society of Microbiology Australian Society for Microbiology WORK EXPERIENCE Research Scientist : SINGHEALTH Research Facilities Neuroscience laboratory Blk A,#02-02, Hospital Drive 169611, Singapore (June 2005- present) Research Assistant : National University of Singapore Microbiology Dept., Faculty of Medicine Science Drive 2, 117597, Singapore March 2005- May 2005 Teaching Assistant : National University of Singapore Microbiology Dept., Faculty of Medicine Science Drive, 117597, Singapore (June 2001-December 2004) Course Tutor : National University of Singapore Biochemistry Dept., Faculty of Medicine Science Drive 2, 117597, Singapore Demystifying the New Genetics (June 2001-June 2002) Teaching Assistant : The Australian National University School of Biochemistry and Molecular Biology Faculty of Science, Canberra 0200 ACT (March 2000- February 2001) Assistant Professorial Lecturer : De La Salle University, College of Science Biology Department, Microbiology 2401 Taft Avenue, 1004, Manila Philippines (May 1992 - October 1993; January 1998-January 1999) 196 Laboratory Specialist : Saudi Arabia National Guard Medical Services King Fahad National Guard Hospital Laboratory Dept. P.O. Box 22490, Riyadh 11426, K.S.A. (October 1993 - September 1997) Senior Microbiologist : King Saud University, College of Medicine Dept. of Clinical Microbiology and Parasitology Asir Central Hospital, P.O. Box 641, Abha, K.S.A. (March 1991 - February 1992) Medical Technologist : U.S. Naval Medical Research Unit-2 San Lazaro Hospital, Microbiology & Clinical Laboratory American Embassy, 1201Roxas Blvd., Manila (February 1989 - January 1991) Lecturer/ Instructor (Part-Time) : Perpetual Help Medical Center & College of Medicine Department of Human Biochemistry and Nutrition Perpetual Help Medical Center, Biñan, Laguna (June 1988 - March 1990) Laboratory Technician : Dr. Annilie Arcangel Clinic and Laboratory 1381 Quiricada Street, Sta. Cruz, Manila (November 1987 - January 1989) RECENT POSTERS Angeles Dario, Chang Siao Yun and Song Keang Peng. “Characterization of Clostridium difficile actin-specific binary ADP-ribosylating toxin detected through genomic DNA products derived from subtractive hybridization between a pathogenic and non-pathogenic strain”. 103rd ASM General Meeting, May 18-22, 2003, Washington Convention Center, Washington D.C. (ASM Student Travel Awardee). Angeles Dario and Song Keang Peng. "Activation of colonic stress signal pathway through Clostridium difficile CDT toxin-Induced ADP-ribosylation and universal shift in actin dynamics". 8th National University of Singapore-National University Hospital Annual Scientific Meeting, Oct. 7-9, 2004. Clinical Research Center Auditorium, Faculty of Medicine. (Best Basic Science Poster, shortlisted). Angeles Dario, Song Keang Peng and Naresh Verma. “The role of serotype-converting temperate bacteriophage SFV genome-encoded factors in Shigella flexneri cell invasion”. 4th Combined Scientific Meeting BRETSS/SSBMB/SSMB, Jan. 15-18, 2003, Raffles Convention Center, Singapore. (Poster Merit Prize, shortlisted). RESEARCH & PUBLICATIONS Angeles, D.C. and Song, K.P. 2005. Peptide antibiotic and actin-binding protein as mixed-type inhibitors of Clostridium difficile CDT toxin activities. Biochemical and Biophysical Research Communications. 327: 361-370. 197 Angeles, D.C. 2005. Cytophysiologic effects and molecular inhibition of a functional actinspecific ADP-ribosylstransferase cdt from Clostridium difficile. Department of Microbiology, Faculty of Medicine, National University of Singapore. Doctoral thesis for the degree: Doctor of Philosophy in Microbiology. In progress. Angeles, D.C. and Song, K.P. 2005. Clostridium difficile CDT toxin-induced ADP-ribosylation of actin distinctly activates colonic stress signal pathway. Research in Microbiology. Submitted. Angeles, D. C., Leong, W.Y., Koay, E., and Song, K.P. 2004. Comparative and quantitative transcription analyses of the binary ADP-ribosyltransferase cdt from a variant Clostridium difficile strain with truncated pathogenicity locus. Journal of Infectious Diseases. Submitted. Angeles, D.C., Ng, K.Y., and Song K.P. 2005. Comparative proteome analysis of Clostridium difficile subcellular fraction and characterization of membrane-associated TcdE of the pathogenicity locus. Manuscript in preparation. Allison, G.E., Angeles, D.C., Huan, P. and Verma, N.K. 2003. Morphology of temperate bacteriophage SFV and characterization of the DNA packaging and capsid genes: The structural genes evolved from two different phage families. The complete genomic sequence of SfV, a serotype-converting temperate bacteriophage of Shigella flexneri. Virology. 308(1): 114-127. Allison, G.E., Angeles, D.C., and Verma, N.K. 2002. The complete genomic sequence of SfV, a serotype-converting temperate bacteriophage of Shigella flexneri. Journal of Bacteriology. 184(7): 1974-1987. Angeles, D.C. 2001. Molecular characterization of the serotype-converting Shigella flexneri bacteriophage SfV. School of Biochemistry and Molecular Biology, The Australian National University. Masteral thesis for the degree: Master of Philosophy in Biochemistry and Molecular Biology. Angeles, D.C., & Montejo, M.M. 2001. Genomic DNA sequencing and physical mapping of Shigella flexneri serotype-specific antigen V temperate bacteriophage. Manila Journal of Science, Agham. 115: 24-36. Angeles, D.C. 1995. Comparison of plasmid profile analysis and antimicrobial resistance pattern in characterizing enterotoxigenic Escherichia coli. Manila Journal of Science, Agham. 19: 37-47. Angeles, D.C., 1994. Molecular characterization of enterotoxigenic Escherichia coli (Philippine isolates) by plasmid DNA and restriction endonuclease fingerprinting. Manila Journal of Science, Agham. 17: 1-7. Angeles, D.C., & Bautista, S. 1993. Use of alkaline phosphatase conjugated oligonucleotide probe for the detection of enterotoxigenic Escherichia coli in fecal samples. Manila Journal of Science, Agham, 16: 7-11. Angeles, D.C., 1992. Detection of heat-stable toxin producing ETEC from watery and formed stools using DNA hybridization and the infant mouse assay. Department of Medical 198 Microbiology, College of Public Health, University of the Philippines. Masteral thesis for the degree: Master of Science in Public Health major in Medical Microbiology. Angeles, D.C. 1987. Effects of high caffeine doses on chromosomal aberrations in mice. Dept., of Biological Sciences, Institute of Biology, The University of the Philippines. Undergraduate thesis for the degree: Bachelor of Science in Biology. REFEREES Tan Eng King, M.D. Associate Professor Singapore General Hospital Neurology Department SINGHEALTH Research Facilities Neurology Laboratory Blk A, #02-02, Hospital Drive 169611, Singapore E-mail: gnrtek@sgh.com.sg Song Keang Peng, Ph.D. Assistant Professor National University of Singapore Microbial Pathogenesis Laboratory Microbiology Dept., Faculty of Medicine Science Drive 2, 117597, Singapore Tel: (65) 6874-3278, Fax: (65) 6776-6872 E-mail: micskp@nus.edu.sg Naresh K. Verma, Ph.D. Associate Professor The Australian National University School of Biochemistry and Molecular Biology Canberra, 0200, ACT, Australia Tel: (612) 6249-2666, Fax: (612) 6249-0313 E-mail: naresh.verma@anu.edu.au Kevin S.W. Tan, Ph.D. Assistant Professor National University of Singapore Lab. of Molecular & Cellular Parasitology Microbiology Dept., Faculty of Medicine Science Drive 2, 117597, Singapore Tel: (65) 6874-6780, Fax: (65) 6776-6872 E-mail: mictank@nus.edu.sg Adelwisa Ortega, M.D., Ph.D. Chairman, Professor University of the Philippines Institute of Public Health Dept. of Medical Microbiology 625 Pedro Gil Ermita, Manila, Philippines Tel: (632) 525-5874 E-mail: Aortega@lagundi.cph.upm.edu.ph Esperanza C. Cabrera, Ph.D. Chairman, Professor De La Salle University College of Science Biology Department, Microbiology 2401 Taft Ave.,1004, Manila, Philippines Tel:(632)524-4611 ext 460; 5360228 199 Fax: (632) 524-0451 E-mail: cosecc@mail.dlsu.edu.ph Stephen E. Walz, Ph.D. Principal Scientist U.S. Naval Medical research Unit-2 NAMRU-2, Box 3, APO AP, CA 96520 U.S.A. Tel: (301) 626-8632 Jocelyn Andrada Secretary to the Counselor for Public Affairs U.S. Information Service American Embassy 1201 Roxas Blvd. Manila, Philippines Tel: (632) 523-1326 E-mail: jmamani@usia.gov Naser El Din Bilal, M.D. Professor King Saud University College of Medicine Dept. of Clinical Microbiology & Parasitology P.O. Box 641, Abha, K.S.A. Tel: (966-07) 224-7800 Manuel J. Padilla, MSMT Medical Laboratory Supervisor Saudi Arabia National Guard Medical Services SANG Health Affairs P.O. Box 8994, Riyadh 11492 KSA Tel: (966-01) 482-9641 ext 409 laboratory Theresita A. Lariosa, M.D. Executive Dean University of Perpetual help & Medical Center Dept. of Clinical Biochemistry & Nutrition Sto.Nino, Binan, Laguna, Philippines Tel: (63) 495118636 ext Col of Medicine LABORATORY DUTIES: (Research/Clinical) • Gene cloning, GST, GFP, 6X His, myc, HA fusion tags • Genome, DNA, RNA extraction • Plasmid and cosmid library production and screening • Site-directed mutagenesis, PCR restriction site linker, Gene-tailor, transposon insertion, SiRNA knock outs • Subtractive hybridization-biotin streptavidin extraction • Colony blot, Southern, Northern, Western hybridization • Genomic and plasmid extraction and purification • Bacteriophage isolation and genome extraction-Induction, PEG purification, dialysis, CsCl ethidium bromide gradient centrifugation. • DNA processing and manipulation: transposon insertion, DNA modification, RT, RealTime PCR quantitation • • • • • • Protein overexpression, Protein purification, affinity chromatography SDS-PAGE – silver stain, coommassie blue stain, ponceau S stain 2-D gel electrophoresis Yeast two hybrid Co-immunoprecipitation 200 • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • Phage plaque assay and sensitivity assay Single and multiplex ELISA microplate assay Western Immunoblotting and hybridization, acetone powder antisera adsorption PCR (Perkin Elmer, BioRad, Hybaid)- colony, gene amplification, sequencing Competent cell prep.- rubidium chloride, calcium chloride, electrocompetent cells Electron microscopy-transmission EM (Hitachi JOELX-200) Confocal microscopy Immunogold labelling DNA sequencing Big dye terminator (ABI prism) and bioinformatics analysis (DNA star, WebANGIS, NCBI program) Protein Sequencing Edman degradation Polyclonal monospecific Antibody production Tissue culture-splitting, passage, feeding Vero, HeLa, McCoy, BHK, Hep-2, Chang Conjunctiva, HCT 116, N1E-115, HepG2 Lyophilization HeLa cell invasion assay Indirect, direct FA- VZ, Mycoplasma, anti-DNA, HSV, RSV, Chlamydia, Leishmania, HIV, HCV, anti-HBc, Hbe, rotavirus Flow cytometry-FACS, T4,T8 ratio FA staining Hemagglutination and indirect hemagglutination assay- Toxoplasma, Schistosoma, Amoeba, Echinococcus, Chikungunya, Rubella, Dengue hemorrhagic, JapB encephalitis Embryonated egg technique-influenza Single radial immunodiffusion-complement and immunoglobulin detection Immunohistochemistry Protein fusion and expression, Western hybridization Radioactive works (3H, 32P, 14C), photoaffinity labeling, ADP-ribosylation assay Phosphorimaging (Typhoon, Amersham) Liquid scintillation counting (MicroBeta, PerkinElmer) Routine microbiological isolation, morphological, biochemical, serological identification Fluorescent microscopy Routine urinalysis (Urotron RL9), fecalysis Hematology: complete blood count, differential count, coagulation tests:prothrombin, thromboplastin, fibrinogen, thrombin/bleeding/clotting time, fibrin degradation, ESR, reticulocyte count, sickle cell, L.E.prep, (Coulter STKS/T890, Cell Dyne 400, Diagnostica Stago Chemistry: serum level of biochemical metabolites-RBS, FBS, BUN, creatinine, cholesterol, triglyceride, AST, ALT, alk phosphatase, etc. (Gemeni, Reflotron, Hitachi 911, Kodak Ectachem, Johnson & Johnson 750 xrc). Endocrinology-LH, FSH, prolactin, cortisol, testosterone, estradiol, ammonia, Mg, Fe,C3, TIBC, etc. (Cobas Mira/DuPont Dimension). Tumor markers- CEA/colon, AFP/liver, MCA/breast, β-HCG/chorion, PSA/prostate, CA19-9/pancreas, CA-15-3/breast, CA125/ovary, etc. Drug assay. Serology: thyroid tests-T3, T4, FT1, 3, 4, TSH,etc (Cobas core, Bm Es300) Flame photometry Histology: H&E staining 201 [...]... A. , Ahmad, M F., Avruch, J., and Woodgett, J R (1994) The stress-activated protein kinase subfamily of c-Jun kinases Nature 369, 156 -160 Lachumanan, R., Armugam, A. , Durairaj, P., Gopalakrishnakone, P., Tan, C H., and Jeyaseelan, K (1999) In situ hybridization and immunohistochemical analysis of the expression of cardiotoxin and neurotoxin genes in Naja naja sputatrix J Histochem Cytochem 47, 55 1 -56 0... 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