This automated method bypasses the need for manual plate reading thus eliminating human bias and error. The image data is translated by LI-COR’s Odyssey software into numerical data which is used directly in the virus titer calculations. (C) 2010 Elsevier B.V. All rights reserved.”
“BACKGROUND

Arterial

calcifications are associated with increased cardiovascular risk, but the genetic basis of this association is unclear.

METHODS

We performed clinical, radiographic, and genetic studies in three families with symptomatic arterial calcifications. Single-nucleotide-polymorphism analysis, targeted gene Selleck AMN-107 sequencing, quantitative polymerase-chain-reaction assays, Western blotting, enzyme measurements, transduction rescue experiments, and in vitro

calcification assays were performed.

RESULTS

We identified nine persons with calcifications of the lower-extremity arteries and hand and foot joint capsules: all five siblings in one family, three siblings in another, and one patient in a third family. Serum calcium, phosphate, and vitamin D levels were normal. Affected members of Family 1 shared a single 22.4-Mb region of homozygosity on chromosome 6 and had a homozygous nonsense mutation (c.662C -> A, p.S221X) in NT5E, encoding CD73, which converts AMP to adenosine. Affected members of Family 2 had a homozygous missense mutation (c.1073G -> A, p.C358Y) in NT5E. The proband of Family 3 was a compound heterozygote for c.662C -> A and c.1609dupA (p.V537fsX7). All mutations found in the three families result in nonfunctional CD73. Cultured fibroblasts from affected members of Family www.selleckchem.com/products/AZD0530.html 1 showed markedly reduced expression of NT5E messenger RNA, CD73 protein, and enzyme activity, as well as increased alkaline phosphatase levels and accumulated calcium phosphate crystals. Genetic rescue experiments normalized the CD73 and alkaline phosphatase activity in patients’ cells, and adenosine treatment reduced the levels of alkaline phosphatase and calcification.

CONCLUSIONS

We identified mutations in NT5E

in members of three families with symptomatic arterial and joint calcifications. This gene encodes CD73, see more which converts AMP to adenosine, supporting a role for this metabolic pathway in inhibiting ectopic tissue calcification.”
“A quantitative real-time reverse transcriptase PCR (RT-qPCR) assay was developed for the analysis of influenza A virus transcription and replication dynamics in mammalian cell culture. The assay is based on a polarity- and sequence-specific reverse transcription used to distinguish specifically between viral genomes (vRNA(-)), replicative intermediates (cRNA(+)) and viral messenger RNAs (vmRNA(+)) of segments 4 (HA), 6 (NA), 7 (M) and 8 (NS) during the life cycle of influenza virus. Synthetic viral RNAs used as reference standards for validation and quantitation were prepared for each viral RNA type and segment.