Ambiguous Base Pairing of 1-(2-Deoxy-β-D-Ribofuranosyl)imidazole-4-carboxamide During PCR

Abstract

The use of 5′-triphosphate of 1-(2-deoxy-β-D-ribofuranosyl)imidazole-4-carboxamide (dYTP) in DNA amplification reaction in place of dATP or dGTP yielded mutations frequencies of 3–4×10^?2 per base per amplification. A reasonable proportion of transversions (11–15%) was observed in the absence of deletions and insertions.     

Purine Nucleoside Phosphorylase Deficiency: A Mutation Update

Purine nucleoside phosphorylase (PNPase) deficiency is an autosomal recessive disorder affecting purine degradation and salvage pathways. Clinically, patients typically present with severe immunodeficiency, neurological dysfunction, and autoimmunity. Biochemically, PNPase deficiency may be suspected in the presence of hypouricemia. We report biochemical and genetic data on a cohort of seven patients from six families identified as PNPase deficient. In all patients, inosine, deoxyinosine, guanosine, and deoxyguanosine were elevated in urine, and mutation analysis revealed seven different mutations of which three were novel. The mutation c.770A>G resulted in the substitution p.His257Arg. A second novel mutation c.257A>G (p.His86Arg) was identified in two siblings and a third novel mutation, c.199C>T (p.Arg67X), was found in a 2-year-old female with delayed motor milestones and recurrent respiratory infections. A review of the literature identified 67 cases of PNPase deficiency from 49 families, including the cases from our own laboratory. PNPase deficiency was confirmed in 30 patients by genotyping and 24 disease causing mutations, including the three novel mutations described in this paper, have been reported to date. In five of the seven patients, plasma uric acid was found to be within the pediatric normal range, suggesting that PNPase deficiency should not be ruled out in the absence of hypouricemia.     

High Frequency Ultrasound for In Vivo Pregnancy Diagnosis and Staging of Placental and Fetal Development in Mice

Background

Ultrasound is a valuable non-invasive tool used in obstetrics and gynecology to monitor the growth and well being of the human fetus. The laboratory mouse has recently emerged as an appropriate model for fetal and perinatal studies because morphogenetic processes in mice exhibit adequate homology to those in humans, and genetic manipulations are relatively simple to perform in mice. High-frequency ultrasound (HFUS) has recently become available for small animal preclinical imaging and can be used to study pregnancy and development in the mouse. The objective of the current study was to assess the main applications of HFUS in the evaluation of fetal growth and placental function and to better understand human congenital diseases.

Methodology/Principal Findings

On each gestational day, at least 5 dams were monitored with HFUS; a total of ∼200 embryos were examined. Because it is not possible to measure each variable for the entire duration of the pregnancy, the parameters were divided into three groups as a function of the time at which they were measured. Univariate analysis of the relationship between each measurement and the embryonic day was performed using Spearman’s rank correlation (Rs). Continuous linear regression was adopted for multivariate analysis of significant parameters. All statistical tests were two-sided, and a p value of 0.05 was considered statistically significant.

Conclusions/Significance

The study describes the main applications of HFUS to assess changes in phenotypic parameters in the developing CD1 mouse embryo and fetus during pregnancy and to evaluating physiological fetal and placental growth and the development of principal organs such as the heart, kidney, liver, brain and eyes in the embryonic mouse. A database of normal structural and functional parameters of mouse development will provide a useful tool for the better understanding of morphogenetic and cardiovascular anomalies in transgenic and mutant mouse models.     

Population Structure of Barley Landrace Populations and Gene-Flow with Modern Varieties

Landraces are heterogeneous plant varieties that are reproduced by farmers as populations that are subject to both artificial and natural selection. Landraces are distinguished by farmers due to their specific traits, and different farmers often grow different populations of the same landrace. We used simple sequence repeats (SSRs) to analyse 12 barley landrace populations from Sardinia from two collections spanning 10 years. We analysed the population structure, and compared the population diversity of the landraces that were collected at field level (population). We used a representative pool of barley varieties for diversity comparisons and to analyse the effects of gene flow from modern varieties. We found that the Sardinian landraces are a distinct gene pool from those of both two-row and six-row barley varieties. There is also a low, but significant, mean level and population-dependent level of introgression from the modern varieties into the Sardinian landraces. Moreover, we show that the Sardinian landraces have the same level of gene diversity as the representative sample of modern commercial varieties grown in Italy in the last decades, even within population level. Thus, these populations represent crucial sources of germplasm that will be useful for crop improvement and for population genomics studies and association mapping, to identify genes, loci and genome regions responsible for adaptive variations. Our data also suggest that landraces are a source of valuable germplasm for sustainable agriculture in the context of future climate change, and that in-situ conservation strategies based on farmer use can preserve the genetic identity of landraces while allowing adaptation to local environments.     

Ultra-Deep Sequencing of Intra-host Rabies Virus Populations during Cross-species Transmission

One of the hurdles to understanding the role of viral quasispecies in RNA virus cross-species transmission (CST) events is the need to analyze a densely sampled outbreak using deep sequencing in order to measure the amount of mutation occurring on a small time scale. In 2009, the California Department of Public Health reported a dramatic increase (350) in the number of gray foxes infected with a rabies virus variant for which striped skunks serve as a reservoir host in Humboldt County. To better understand the evolution of rabies, deep-sequencing was applied to 40 unpassaged rabies virus samples from the Humboldt outbreak. For each sample, approximately 11 kb of the 12 kb genome was amplified and sequenced using the Illumina platform. Average coverage was 17,448 and this allowed characterization of the rabies virus population present in each sample at unprecedented depths. Phylogenetic analysis of the consensus sequence data demonstrated that samples clustered according to date (1995 vs. 2009) and geographic location (northern vs. southern). A single amino acid change in the G protein distinguished a subset of northern foxes from a haplotype present in both foxes and skunks, suggesting this mutation may have played a role in the observed increased transmission among foxes in this region. Deep-sequencing data indicated that many genetic changes associated with the CST event occurred prior to 2009 since several nonsynonymous mutations that were present in the consensus sequences of skunk and fox rabies samples obtained from 20032010 were present at the sub-consensus level (as rare variants in the viral population) in skunk and fox samples from 1995. These results suggest that analysis of rare variants within a viral population may yield clues to ancestral genomes and identify rare variants that have the potential to be selected for if environment conditions change.     

Inhibition of Histo-blood Group Antigen Binding as a Novel Strategy to Block Norovirus Infections

Noroviruses (NoVs) are the most important viral pathogens that cause epidemic acute gastroenteritis. NoVs recognize human histo-blood group antigens (HBGAs) as receptors or attachment factors. The elucidation of crystal structures of the HBGA-binding interfaces of a number of human NoVs representing different HBGA binding patterns opens a new strategy for the development of antiviral compounds against NoVs through rational drug design and computer-aided virtual screening methods. In this study, docking simulations and virtual screening were used to identify hit compounds targeting the A and B antigens binding sites on the surface of the capsid P protein of a GII.4 NoV (VA387). Following validation by re-docking of the A and B ligands, these structural models and AutoDock suite of programs were used to screen a large drug-like compound library (derived from ZINC library) for inhibitors blocking GII.4 binding to HBGAs. After screening >2 million compounds using multistage protocol, 160 hit compounds with best predicted binding affinities and representing a number of distinct chemical classes have been selected for subsequent experimental validation. Twenty of the 160 compounds were found to be able to block the VA387 P dimers binding to the A and/or B HBGAs at an IC₅₀<40.0 µM, with top 5 compounds blocking the HBGA binding at an IC₅₀<10.0 µM in both oligosaccharide- and saliva-based blocking assays. Interestingly, 4 of the top-5 compounds shared the basic structure of cyclopenta [a] dimethyl phenanthren, indicating a promising structural template for further improvement by rational design.