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Methods: Twenty patients with abdominal aortic aneurysms were investigated before elective surgery with a 1.5 Tesla MR-scanner. Standard multislice spin-echo sequences were used for aneurysm imaging. A flow-adjusted gradients sequence (FLAG) provided three-dimensional vector plots depicting local blood flow velocities as functions of time and anatomical position. Computer-generated animated presentations of the vectors were developed to ease data analysis and interpretation.

All plasmids were purified using JETstar2.0 mini and maxi-prep Kit (GenoMed, St. Louis, Missouri, USA) according to the manufacturer's instructions. Pf 3D7 small-scale gDNA isolation followed the MR4 protocol. The HNF-1 cDNA clone was obtained from Open Biosystems (Accession No: {"type":"entrez-nucleotide","attrs":{"text":"BC104908","term_id":"85396995","term_text":"BC104908"}}BC104908; Rockford, Illinois, USA) and was amplified by PCR with primers F (5'-ATAGAA TTCGCAGCCGAGCCATGGTTTCTA-3'; EcoRI site underlined) and R (5'-ATACTCGAGTGCCGTGGTTACTGGGAGGAAG-3'; XhoI site underlined). The amplified fragment was inserted into pCDNA3-Flag-HA (Addgene, Cambridge, Massachusetts, USA) by EcoRI and XhoI ligation. Lentivirus (LV) HNF-1 was constructed by cutting the LV plasmid backbone with BamHI and XbaI to expel the resident luciferase gene, which was replaced with a HNF-1 cDNA insert cut from pCDNA3-HNF-1-Flag-HA. A construct for luciferase activity was based on a pGL3-Basic vector (Promega, Madison, Wisconsin, USA). Two different 5'-upstream sequences flanking the Pf-Sub2 gene were amplified from Pf 3D7 gDNA with primers. The primers used for -2,567/+32 UTR were F (5'-ATAGGTACCTAGAATAGAATAAAATAAATAACC-3'; KpnI site underlined) and R (5'-ATAAAGCTTATCAAGGAAACCACATAA-3'; HindIII site underlined). The primers used for -1,422/+12 UTR were F (5'-AAGGTACCATGGTATGAGTTCTTTATA-3'; KpnI site underlined) and R (5'-ATGCTAGCAATATTCAGCATTATACGGA-3'; NheI site underlined). PCR was performed using a TGRADIENT thermocycler (Biometra, Goettingen, Germany). The amplified UTR sequences were cloned into a pMDT vector (Takara Bio, Shiga, Japan) and subcloned into a pGL3-Basic vector with KpnI and NheI/HindIII sticky end ligation, generating the pGL3-Basic-Pf-Sub2 5'-UTR construct. The other upstream inserts were cloned into a pGL3-Basic vector by blunt restriction sites (EcoRV, SnaBI and SwaI) located in the 1.5-kb region upstream of the luciferase gene. The internal control was pSV--galactosidase (Promega). All clones were analyzed by restriction mapping and were sequenced by a 3730XL DNA sequencer (Macrogen, Seoul, Korea).

To analyze the influence of HNF-1 on the transcriptional activity of 5'-UTR of Pf-Sub2 in vitro, co-transfection of pGL3-Basic-Pf-Sub2 5'-UTR with different amounts of HNF-1 was performed in 293T cells. The exogenous HNF-1 expression level of pCDNA-HNF-1-Flag-HA in transfected 293T cells was ascertained by western blotting with anti-HNF-1 antibody (Fig. 2A). High HNF-1 expression was observed using 4.5 g of the pCDNA-HNF-1-Flag-HA transfection vector. Except for the -329/+12 UTR, the 5'-UTRs showed high and distinct transcriptional luciferase activities (Fig. 2B). As the amount of pCDNA-HNF-1-Flag-HA vector increased from 0.5 to 4.5 g, luciferase activity was increased. Although the -1,422/+12 UTR did not drive the luciferase activity in a similar manner as apparent in Fig. 2A, a significant difference was seen with 1.5 g of pGL3-Basic-Pf-Sub2 5'-UTR/4.5 g of pCDNA-HNF-1-flag-HA as compared with only 1.5 g of pGL3-Basic-Pf-Sub2 5'-UTR.

We thank Dr. Zhang Sizhong for his constructive suggestions, Dr. Didier Trono for kindly providing the lentiviral vector system, and Dr. Peter Mackenzie for the HNF-1 plasmid construct. This research was supported by Wonkwang University in 2010. 006ab0faaa