BACKGROUND: Newborn screening for lysosomal storage diseases (LSDs) has been gaining considerable interest owing to the availability of enzyme replacement therapies. We present a digital microfluidic platform to perform rapid, multiplexed enzymatic analysis of acid α-glucosidase (GAA) and acid α-galactosidase to screen for Pompe and Fabry disorders. The results were compared with those obtained using standard fluorometric methods.METHODS: We performed bench-based, fluorometric enzymatic analysis on 60 deidentified newborn dried blood spots (DBSs), plus 10 Pompe-affected and 11 Fabry-affected samples, at Duke Biochemical Genetics Laboratory using a 3-mm punch for each assay and an incubation time of 20 h. We used a digital microfluidic platform to automate fluorometric enzymatic assays at Advanced Liquid Logic Inc. using extract from a single punch for both assays, with an incubation time of 6 h. Assays were also performed with an incubation time of 1 h.RESULTS: Assay results were generally comparable, although mean enzymatic activity for GAA using microfluidics was approximately 3 times higher than that obtained using bench-based methods, which could be attributed to higher substrate concentration. Clear separation was observed between the normal and affected samples at both 6- and 1-h incubation times using digital microfluidics.CONCLUSIONS: A digital microfluidic platform compared favorably with a clinical reference laboratory to perform enzymatic analysis in DBSs for Pompe and Fabry disorders. This platform presents a new technology for a newborn screening laboratory to screen LSDs by fully automating all the liquid-handling operations in an inexpensive system, providing rapid results.

Background: We used doxorubicin-based chemotherapy as a clinical model of oxidative assault in humans. Methods: The study recruited newly diagnosed breast cancer patients (n = 23). Urine samples were collected immediately before (T0) and at 1 hour (T1) and 24 hours (T24) after i.v. administration of treatment. Measurements included allantoin and the isoprostanes iPF(2α)-III, iPF(2α)-VI, and 8,12-iso-iPF(2α)-VI along with the prostaglandin 2,3-dinor-iPF(2α)-III, a metabolite of iPF(2α)-III. All biomarkers were quantified using liquid chromatography–tandem mass spectrometry. Results: In all subjects, the levels of the biomarkers increased at T1: allantoin by 22% (P = 0.06), iPF(2α)-III by 62% (P < 0.05), iPF(2α)-VI by 41% (P < 0.05), 8,12-iso-iPF(2α)-VI by 58% (P < 0.05), and 2,3-dinor-iPF(2α)-III by 52% (P < 0.05). At T24, the F2-isoprostanes returned to their baseline levels; the levels of allantoin continued to increase, although the T24-T0 difference was not statistically significant. Conclusions: These results indicate that urinary F2-isoprostanes are valid biomarkers and allantoin is a promising biomarker of oxidative status in humans. Impact: The levels of biomarkers change quickly in response to oxidative assault and can be used to monitor oxidative status in humans in response to treatments related either to generation of free radicals (chemotherapy and radiation therapy) or to antioxidants (inborn metabolic diseases and Down syndrome). Cancer Epidemiol Biomarkers Prev; 19(6); 1506–10. ©2010 AACR.

Nucleotide-dependent unblocking of chain-terminated DNA by human immunodeficiency virus type 1 reverse transcriptase (RT) is enhanced by the presence of mutations associated with 3′-azido-3′-deoxythymidine (AZT) resistance. The increase in unblocking activity was greater for mutant combinations associated with higher levels of in vivo AZT resistance. The difference between mutant and wild-type activity was further enhanced by introduction of a methyl group into the nucleotide substrate and was decreased for a nonaromatic substrate, suggesting that π-π interactions between RT and an aromatic structure may be facilitated by these mutations.