DNA damaging effects of benzotriazine-N-oxides

The aim of this study was to investigate the DNA damaging abilities of SR 4233 (Tirapazamine) and a range of congeners. These are novel benzotriazine-N-oxide compounds of potential clinical importance in the treatment of hypoxic tumours. The studies used a viral double transfection assay which involved exposing purified $X174 DNA to the compounds under oxic, hypoxic and hypoxic reductive conditions and subsequently transfecting the DNA into E. coli AB1157 and E. coli C. Experiments under reductive hypoxia were carried out between pH 4-7 to establish if a reduction product required protonation for the DNA damage process. The biologically relevant druginduced DNA damage was assessed by utilising a range of E. coli mutants deficient in specific DNA repair genes, the products of which are involved in excision, recombination and SOS repair. Viscometry was used to assess the effects of various druginucleotide ratios and the ionic strength of the buffer on the DNA damage caused. DNA has been confirmed as a target for the action of the benzotriazine-N-oxides. SR 4233, the lead compound, was found to be the most active of all the compounds tested. Damage was only induced upon reduction of the compounds and there was no significant damage to DNA under oxic or hypoxic conditions. SR 4233 exhibited increased DNA damage at acid pH indicating that the radical anion responsible for DNA damage has a requirement for protonation. The damaging species is probably the 1-electron reduction product of SR 4233 as SR 4317 and SR 4330, the 2-electron and 4-electron reduction products respectively, caused significantly less DNA damage under hypoxic reductive conditions. There is also evidence that the disproportionation reaction, which can lead to radical production without DNA damage, may not be an important reaction for SR 4233 bioactivation. Studies with repair-deficient mutants of E. coli indicated that SR 4233 is capable of inducing DNA damage which is recognised and repaired by the ABC excinuclease complex. However, the major damage caused is recognised and repaired by the gene products of the xth and nth mutants. This indicates that reduced SR 4233 induces primarily pyrimidine based oxidative damage in DNA

Adefovir dipivoxil for wait-listed and post-liver transplantation patients with lamivudine-resistant hepatitis B : final long-term results

Wait-listed (n = 226) or post-liver transplantation (n = 241) chronic hepatitis B (CHB) patients with lamivudine-resistant hepatitis B virus (HBV) were treated with adefovir dipivoxil for a median of 39 and 99 weeks, respectively. Among wait-listed patients, serum HBV DNA levels became undetectable (<1,000 copies/mL) in 59% and 65% at weeks 48 and 96, respectively. After 48 weeks, alanine aminotransferase (ALT), albumin, bilirubin, and prothrombin time normalized in 77%, 76%, 60%, and 84% of wait-listed patients, respectively. Among posttransplantation patients, serum HBV DNA levels became undetectable in 40% and 65% at weeks 48 and 96, respectively. After 48 weeks, ALT, albumin, bilirubin, and prothrombin time normalized in 51%, 81%, 76%, and 56% of posttransplantation patients, respectively. Among wait-listed patients who underwent on-study liver transplantation, protection from graft reinfection over a median of 35 weeks was similar among patients who did (n = 34) or did not (n = 23) receive hepatitis B immunoglobulin (HBIg). Hepatitis B surface antigen was detected on the first measurement only in 6% and 9% of patients who did or did not receive HBIg, respectively. Serum HBV DNA was detected on consecutive visits in 6% and 0% of patients who did or did not receive HBIg, respectively. Treatment-related adverse events led to discontinuation of adefovir dipivoxil in 4% of patients. Cumulative probabilities of resistance were 0%, 2%, and 2% at weeks 48, 96, and 144, respectively. In conclusion, adefovir dipivoxil is effective and safe in wait-listed or posttransplantation CHB patients with lamivudine-resistant HBV and prevents graft reinfection with or without HBIg

Accurate whole human genome sequencing using reversible terminator chemistry

DNA sequence information underpins genetic research, enabling discoveries of important biological or medical benefit. Sequencing projects have traditionally used long (400-800 base pair) reads, but the existence of reference sequences for the human and many other genomes makes it possible to develop new, fast approaches to re-sequencing, whereby shorter reads are compared to a reference to identify intraspecies genetic variation. Here we report an approach that generates several billion bases of accurate nucleotide sequence per experiment at low cost. Single molecules of DNA are attached to a flat surface, amplified in situ and used as templates for synthetic sequencing with fluorescent reversible terminator deoxyribonucleotides. Images of the surface are analysed to generate high-quality sequence. We demonstrate application of this approach to human genome sequencing on flow-sorted X chromosomes and then scale the approach to determine the genome sequence of a male Yoruba from Ibadan, Nigeria. We build an accurate consensus sequence from >30x average depth of paired 35-base reads. We characterize four million single-nucleotide polymorphisms and four hundred thousand structural variants, many of which were previously unknown. Our approach is effective for accurate, rapid and economical whole-genome re-sequencing and many other biomedical applications