Growing evidence suggests that oxidative damage to cells generates mutagenic 7,8-dihydro-8-oxo-2 -deoxyguanosine (8-oxodG), which may initiate diseases related to aging and carcinogenesis. Kinetic measurement of 8-oxodG metabolism and repair in cells has been hampered by poor assay sensitivity and by difficulty characterizing the flux of oxidized nucleotides through the relevant metabolic pathways. We report here the development of a sensitive and quantitative approach to characterizing the kinetics and metabolic sources of 8-oxodG in MCF-7 human breast cancer cells by accelerator mass spectrometry. We observed that [ 14 C]8-oxodG at medium concentrations of up to 2 pmol/ml was taken up by MCF-7 cells, phosphorylated to mono-, di-, and triphosphate derivatives, and incorporated into DNA. Oxidative stress caused by exposure of the cells to 17␤-estradiol resulted in a reduction in the rate of [ 14 C]8-oxodG incorporation into DNA and an increase in the ratio of 8-oxodG monophosphate (8-oxodGMP) to 8-oxodG triphosphate (8-oxodGTP) in the nucleotide pool. 17␤-Estradiolinduced oxidative stress up-regulated the nucleotide pool cleansing enzyme MTH1 and possibly other Nudix-related pyrophosphohydrolases. These data support the conclusion that 8-oxodGTP is formed in the nucleotide pool by both 8-oxodG metabolism and endogenous reactive oxygen species. The metabolism of 8-oxodG to 8-oxodGTP, followed by incorporation into DNA is a mechanism by which the cellular presence of this oxidized nucleoside can lead to mutations.DNA repair ͉ nucleoside metabolism ͉ oxidative stress ͉ breast cancer

Within the last decade new technologies have been developed and implemented which employ light, often in the presence of a photosensitizer, to inactivate pathogens that reside in human blood products for the purpose of transfusion. These pathogen reduction technologies attempt to find the proper balance between pathogen kill and cell quality. Each system utilizes various chemistries that not only impact which pathogens they can inactivate and how, but also how the treatments affect the plasma and cellular proteins and to what degree. This paper aims to present the various chemical mechanisms for pathogen reduction in transfusion medicine that are currently practiced or in development.

The PrepFiler Forensic DNA Extraction Kit enables isolation of genomic DNA from a variety of biological samples. The kit facilitates reversible binding of DNA with magnetic particles resulting in high DNA recovery from samples with very low and high quantities of biological materials: 0.1 and 40 microL of human blood (donor 2) provided 14 and 2883 ng of DNA, respectively. Following the revised SWGDAM guidelines, performance of the developed method was investigated using different sample types including saliva on swabs, semen stains on cotton fabric, samples exposed to environment, samples with polymerase chain reaction (PCR) inhibitors, blood stains (on denim, cotton cloth, and FTA paper), and touch evidence-type samples. DNA yields for all samples tested were equal or better than those obtained by both phenol-chloroform extraction and commercial kits tested. DNA obtained from these samples was free of detectable PCR inhibitors. Short tandem repeat profiles were complete, conclusive, and devoid of PCR artifacts.

A differential sized amplicon real-time PCR assay of mitochondrial DNA effectively documents nucleic acid damage induced by Mirasol treatment of platelets. The use of the assay for plasma product pathogen reduction requires further investigation.

7,8-Dihydro-8-oxo-2′-deoxyguanosine (8-oxodG) is a well-known marker of oxidative stress. We report a mechanistic analysis of several pathways by which 8-oxodG is converted to nucleotide triphosphates and incorporated into both DNA and RNA. Exposure of MCF-7 cells to [14C]8-oxodG combined with specific inhibitors of several nucleotide salvage enzymes followed with accelerator mass spectrometry provided precise quantitation of the resulting radiocarbon-labeled species. Concentrations of exogenously dosed nucleobase in RNA reached one per 106 nucleotides, 5–6-fold higher than the maximum observed in DNA. Radiocarbon incorporation into DNA and RNA was abrogated by Immucillin H, an inhibitor of human purine nucleoside phosphorylase (PNP). Inhibition of ribonucleotide reductase (RR) decreased the radiocarbon content of the DNA, but not in RNA, indicating an important role for RR in the formation of 8-oxodG-derived deoxyribonucleotides. Inhibition of deoxycytidine kinase had little effect on radiocarbon incorporation in DNA, which is in contrast to the known ability of mammalian cells to phosphorylate dG. Our data indicate that PNP and RR enable nucleotide salvage of 8-oxodG in MCF-7 cells, a previously unrecognized mechanism that may contribute to mutagenesis and carcinogenesis.

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The HID EVOlution—Extraction System (Tecan Group Ltd., Mannedorf, Switzerland) was developed to automate DNA extraction from biological samples using the PrepFiler Automated Forensic DNA Extraction Kit (Applied Biosystems, Foster City, CA). The system consists of a Tecan Freedom EVO 150 robot (Tecan Group Ltd., Mannedorf, Switzerland), a graphical user interface designed for use with Freedom EVOware software v 2.1 SPI (Tecan Group Ltd., Mannedorf, Switerland) as well as instrument hardware and plastic to support the PrepFiler reagents and protocol. The DNA quality and quantity obtained were comparable to that observed with the corresponding manual extraction protocol. Purified DNA was free of inhibitors and ready for downstream applications, such as real-time quantitative PCR and PCR for short tandem repeat (STR) analysis. The DNA quantity and quality obtained were consistent as demonstrated by the quantification and STR results. Our studies indicate that the HID EVOlution—Extraction System can easily be adopted in forensic laboratories to alleviate some of the bottlenecks of sample preparation in forensic laboratories.

Background: Sub-Saharan African countries utilize whole blood (WB) to treat severe anemia secondary to severe blood loss or malaria on an emergency basis. In many areas with high prevalence of transfusion-transmissible agents, blood safety measures are insufficient. Pathogen reduction technology applied to WB might considerably improve blood safety. Methods: Whole blood from 40 different donors were treated with riboflavin and UV light (pathogen reduction technology) in order to inactivate malaria parasite replication. The extent of parasite inactivation was determined using quantitative polymerase chain reaction methods and was correlated to studies evaluating the replication of malaria parasites in culture. Products were also stored for 21 days at +4°C and monitored for cell quality throughout storage. Results: Plasmodium amplicon was present in 21 samples (>100 copies/mL), doubtful in four (10–100 genome equivalents [gEq]/mL), and negative in 15 U. The majority of asymptomatic parasitemic donors carried low parasite levels, with only six donors above 5,000 copies/mL (15%). After treatment with riboflavin and UV light, these six samples demonstrated a 0.5 to 1.2 log reduction in quantitative polymerase chain reaction amplification. This correlated to equal to or greater than 6.4 log reductions in infectivity. In treated WB units, cell quality parameters remained stable; however, plasma hemoglobin increased to 0.15 g/dL. All markers behaved similarly to published data for stored, untreated WB. Conclusions: Pathogen reduction technology treatment can inactivate malaria parasites in WB while maintaining adequate blood quality during posttreatment cold storage for 21 days.