- Email: [email protected]
The effect of polymorphisms in CYP2C9, CYP4F2, EPHX1 and VKORC1 on warfarin dose in Turkish patients
Abstracts / Current Opinion in Biotechnology 22S (2011) S15–S152
O4
S125
Optimization in isolation and purification of plasmid DNA (pAcGFP1-N1)
considered as monodisperse. The colloidal properties and stability of commercially available non-viral gene delivery systems should be well-defined before optimizing the parameters in transfection studies.
Turkan Eldem 1 , Pelin Yargan 1 , Ozgen Koseoglu Eser 2
doi:10.1016/j.copbio.2011.05.407
1
Pharmaceutical Biotechnology Department, Faculty of Pharmacy, Hacettepe University, Ankara, Turkey 2 Medical Microbiology Department, Faculty of Medicine, Hacettepe University, Ankara, Turkey E-mail address: [email protected] (T. Eldem) High quality supercoiled pDNA is required for both in vitro and in vivo gene delivery studies. The purpose of this study is to select optimum isolation and purification conditions for pAcGFP1N1, a novel GFP reporter system, with high yield and purity. After transforming Escherichia coli DH5-( (Invitrogen, USA) competent cells by pAcGFP1-N1 (Clontech, USA), cells were diluted with LB medium containing kanamycin in different ratios (1:1000 (BC1); 1:500 (BC2)), cultivated overnight and optical densities were measured. The isolation-purification steps were conducted using Qiagen’s Plasmid Midi Kit and Qiafilter Cartridges (QiagenGermany), whereas modifications were performed in several steps such as on-ice incubation, centrifugation, using elution buffers at room temperature and 65 ◦ C, sequential elutions and isopropanol storage (−20 ◦ C). The yield, purity and integrity were evaluated by spectrophotometer (NanoDrop-1000, Thermo-USA) and electrophoresis respectively. The concentrations determined for BC1 and BC2 were 3.39 × 109 and 3.89 × 109 cells/mL respectively. The yield and purity (A260/280 and A260/230 ) values for pAcGFP1-N1 were 175%, 1.875, 2.150 (BC1) and 151%; 1.870, 2.187 (BC2) respectively and electrophoresis results confirmed the integrity. Maximized pAcGFP1-N1 yields were obtained with high purity only after optimization and application of modifications.
O6 The effect of polymorphisms in CYP2C9, CYP4F2, EPHX1 and VKORC1 on warfarin dose in Turkish patients Mahmut Ozer 1 , Yeliz Demirci 1 , Sabit Sarikaya 2 , Iskender Karalti 5 , Cigdem Kaspar 4 , Candan Hizel 3 , Serdar Alpan 1 , Ece Genc 1 1
Yeditepe University, Pharmacology Department, Turkey Kartal Kosuyolu Education and Research Hospital, Cardiac Surgery Department, Turkey 3 C2H-Vichy Genomics, France 4 Yeditepe University, Biostatistic Department, Turkey 5 Yeditepe University, Clinic Microbiology Department, Turkey 2
E-mail address: [email protected] (M. Ozer)
Pharmaceutical Biotechnology Department, Faculty of Pharmacy, Hacettepe University, Ankara, Turkey
The aim of this study is to determine whether and to what extent the variability in warfarin dose requirements was determined by polymorphisms in CYP2C9, CYP4F2, EPHX1 and VKORC1 by a Turkish population. Patients (n = 100) who had stable doses and (International Normalized Ratio) INRs at their last three consecutive visits were registered. Their concurrent medications, chronic illnesses, warfarin related bleedings or thromboembolisms, smoking, alcohol intake and weekly green vegetable consumption were recorded. Blood samples were drawn, DNA was isolated and genotyped by Real Time-PCR for polymorphisms in target genes. Statistical analysis was used to determine the independent effects of genetic and non-genetic factors on warfarin dose optimization. In addition to age, genetic variants of CYP2C9 and VKORC1 were found to be significant predictor variables for the maintenance dose for warfarin, explaining 28% of dose variability. VKORC1 and CYP2C9 genotypes remain predictor variables of the warfarin dose. Additionally, we confirmed the finding that CYP4F2 also contributes to dose variability. The translation of this knowledge into clinical guidelines can be useful for the adjustment of the initial warfarin dose, reduction of the burden of frequent INR measurements and improvement of the safe use of this popular anticoagulant by Turkish patients.
E-mail address: [email protected] (P. Yargan)
doi:10.1016/j.copbio.2011.05.408
doi:10.1016/j.copbio.2011.05.406
O5 Colloidal properties of Lipofectamine 2000 by dynamic light scattering (DLS) Pelin Yargan, Turkan Eldem
To evaluate the particle size analysis of Lipofectamine 2000 (LF2000) by DLS, since LF2000 is a widely used cationic liposomal agent in transfection studies and there is a lack of information about its colloidal properties during the shelf-life. The DLS measurements of LF2000 (Invitrogen-USA) independent samples in cell culture water (CCW) (Biological Industries, Israel), water for injection (WPI) (I.Ethem, Turkiye) and PBS pH = 7.5 (Amresco, USA) were performed on a Zetasizer Nano-S (Malvern Instruments, UK) at 25 ◦ C using general purpose analysis (5 minutes equilibration; measurement duration:30 seconds, 10 runs). The intensity weighted mean hydrodynamic size expressed as the Z-average (nm) and the polydispersity index (PDI) (mean ± standard error, n = 15) were evaluated by ANOVA. The Z-average and the PDI of LF2000 samples in CCW, WFI and PBS were 111 ± 5.51 nm and 0.285 ± 0.019, 100 ± 1.62 nm and 0.253 ± 0.016, 630 ± 23.2 nm and 0.202 ± 0.011 having secondary peaks between 259 and 5171 nm, 1428 and 5186 nm, 5061 and 5505 nm respectively. Due to the mid-range polydispersity observed, the liposomal size of LF2000 cannot be
O7 Producing the recombinant CTB as a delivery/targeting vehicle Zeinab Karimi, Mohammad Bagher Ghoshoun, Narjes Ebrahimi, Ahmad Gholami, Abdollah Ghasemian, Younes Ghasemi Pharmaceutical Biotechnology, Faculty of Pharmacy, Shiraz University of Medical Sciences, Shiraz, Iran E-mail address: [email protected] (Z. Karimi) B subunit of Vibrio cholerae toxin (CTB) has been studied as a protein with GM-1 binding, immunomodulatory and carrier activity. Designing of CTB mutant form as a targeting delivery vehicle V. cholerae CTB coding gene was amplified by using specific designed primers. The amplified DNA sequence was cloned into expression vector, pET22b, followed by confirmation through PCR, restriction
O4
S125
Optimization in isolation and purification of plasmid DNA (pAcGFP1-N1)
considered as monodisperse. The colloidal properties and stability of commercially available non-viral gene delivery systems should be well-defined before optimizing the parameters in transfection studies.
Turkan Eldem 1 , Pelin Yargan 1 , Ozgen Koseoglu Eser 2
doi:10.1016/j.copbio.2011.05.407
1
Pharmaceutical Biotechnology Department, Faculty of Pharmacy, Hacettepe University, Ankara, Turkey 2 Medical Microbiology Department, Faculty of Medicine, Hacettepe University, Ankara, Turkey E-mail address: [email protected] (T. Eldem) High quality supercoiled pDNA is required for both in vitro and in vivo gene delivery studies. The purpose of this study is to select optimum isolation and purification conditions for pAcGFP1N1, a novel GFP reporter system, with high yield and purity. After transforming Escherichia coli DH5-( (Invitrogen, USA) competent cells by pAcGFP1-N1 (Clontech, USA), cells were diluted with LB medium containing kanamycin in different ratios (1:1000 (BC1); 1:500 (BC2)), cultivated overnight and optical densities were measured. The isolation-purification steps were conducted using Qiagen’s Plasmid Midi Kit and Qiafilter Cartridges (QiagenGermany), whereas modifications were performed in several steps such as on-ice incubation, centrifugation, using elution buffers at room temperature and 65 ◦ C, sequential elutions and isopropanol storage (−20 ◦ C). The yield, purity and integrity were evaluated by spectrophotometer (NanoDrop-1000, Thermo-USA) and electrophoresis respectively. The concentrations determined for BC1 and BC2 were 3.39 × 109 and 3.89 × 109 cells/mL respectively. The yield and purity (A260/280 and A260/230 ) values for pAcGFP1-N1 were 175%, 1.875, 2.150 (BC1) and 151%; 1.870, 2.187 (BC2) respectively and electrophoresis results confirmed the integrity. Maximized pAcGFP1-N1 yields were obtained with high purity only after optimization and application of modifications.
O6 The effect of polymorphisms in CYP2C9, CYP4F2, EPHX1 and VKORC1 on warfarin dose in Turkish patients Mahmut Ozer 1 , Yeliz Demirci 1 , Sabit Sarikaya 2 , Iskender Karalti 5 , Cigdem Kaspar 4 , Candan Hizel 3 , Serdar Alpan 1 , Ece Genc 1 1
Yeditepe University, Pharmacology Department, Turkey Kartal Kosuyolu Education and Research Hospital, Cardiac Surgery Department, Turkey 3 C2H-Vichy Genomics, France 4 Yeditepe University, Biostatistic Department, Turkey 5 Yeditepe University, Clinic Microbiology Department, Turkey 2
E-mail address: [email protected] (M. Ozer)
Pharmaceutical Biotechnology Department, Faculty of Pharmacy, Hacettepe University, Ankara, Turkey
The aim of this study is to determine whether and to what extent the variability in warfarin dose requirements was determined by polymorphisms in CYP2C9, CYP4F2, EPHX1 and VKORC1 by a Turkish population. Patients (n = 100) who had stable doses and (International Normalized Ratio) INRs at their last three consecutive visits were registered. Their concurrent medications, chronic illnesses, warfarin related bleedings or thromboembolisms, smoking, alcohol intake and weekly green vegetable consumption were recorded. Blood samples were drawn, DNA was isolated and genotyped by Real Time-PCR for polymorphisms in target genes. Statistical analysis was used to determine the independent effects of genetic and non-genetic factors on warfarin dose optimization. In addition to age, genetic variants of CYP2C9 and VKORC1 were found to be significant predictor variables for the maintenance dose for warfarin, explaining 28% of dose variability. VKORC1 and CYP2C9 genotypes remain predictor variables of the warfarin dose. Additionally, we confirmed the finding that CYP4F2 also contributes to dose variability. The translation of this knowledge into clinical guidelines can be useful for the adjustment of the initial warfarin dose, reduction of the burden of frequent INR measurements and improvement of the safe use of this popular anticoagulant by Turkish patients.
E-mail address: [email protected] (P. Yargan)
doi:10.1016/j.copbio.2011.05.408
doi:10.1016/j.copbio.2011.05.406
O5 Colloidal properties of Lipofectamine 2000 by dynamic light scattering (DLS) Pelin Yargan, Turkan Eldem
To evaluate the particle size analysis of Lipofectamine 2000 (LF2000) by DLS, since LF2000 is a widely used cationic liposomal agent in transfection studies and there is a lack of information about its colloidal properties during the shelf-life. The DLS measurements of LF2000 (Invitrogen-USA) independent samples in cell culture water (CCW) (Biological Industries, Israel), water for injection (WPI) (I.Ethem, Turkiye) and PBS pH = 7.5 (Amresco, USA) were performed on a Zetasizer Nano-S (Malvern Instruments, UK) at 25 ◦ C using general purpose analysis (5 minutes equilibration; measurement duration:30 seconds, 10 runs). The intensity weighted mean hydrodynamic size expressed as the Z-average (nm) and the polydispersity index (PDI) (mean ± standard error, n = 15) were evaluated by ANOVA. The Z-average and the PDI of LF2000 samples in CCW, WFI and PBS were 111 ± 5.51 nm and 0.285 ± 0.019, 100 ± 1.62 nm and 0.253 ± 0.016, 630 ± 23.2 nm and 0.202 ± 0.011 having secondary peaks between 259 and 5171 nm, 1428 and 5186 nm, 5061 and 5505 nm respectively. Due to the mid-range polydispersity observed, the liposomal size of LF2000 cannot be
O7 Producing the recombinant CTB as a delivery/targeting vehicle Zeinab Karimi, Mohammad Bagher Ghoshoun, Narjes Ebrahimi, Ahmad Gholami, Abdollah Ghasemian, Younes Ghasemi Pharmaceutical Biotechnology, Faculty of Pharmacy, Shiraz University of Medical Sciences, Shiraz, Iran E-mail address: [email protected] (Z. Karimi) B subunit of Vibrio cholerae toxin (CTB) has been studied as a protein with GM-1 binding, immunomodulatory and carrier activity. Designing of CTB mutant form as a targeting delivery vehicle V. cholerae CTB coding gene was amplified by using specific designed primers. The amplified DNA sequence was cloned into expression vector, pET22b, followed by confirmation through PCR, restriction