A gene-driven ENU-based approach to generating an allelic series in any gene

N-ethyl-N-nitrosourea (ENU) introduces mutations throughout the mouse genome at relatively high efficiency. Successful high-throughput phenotype screens have been reported and alternative screens using sequence-based approaches have been proposed. For the purpose of generating an allelic series in selected genes by a sequence-based approach, we have constructed an archive of over 4000 DNA samples from individual F₁ ENU-mutagenized mice paralleled by frozen sperm samples. Together with our previously reported archive, the total size now exceeds 6000 individuals. A gene-based screen of 27.4 Mbp of DNA, carried out using denaturing high-performance liquid chromatography (DHPLC), found a mutation rate of 1 in 1.01 Mbp of which 1 in 1.82 Mbp were potentially functional. Screening of whole or selected regions of genes on subsets of the archive has allowed us to identify 15 new alleles from 9 genes out of 15 tested. This is a powerful adjunct to conventional mutagenesis strategies and has the advantage of generating a variety of alleles with potentially different phenotypic outcomes that facilitate the investigation of gene function. It is now available to academic collaborators as a community resource.     

Conversion of array‐based single nucleotide polymorphic markers for use in targeted genotyping by sequencing in hexaploid wheat (Triticum aestivum)

Wheat breeders and academics alike use single nucleotide polymorphisms (SNP s) as molecular markers to characterize regions of interest within the hexaploid wheat genome. A number of SNP ‐based genotyping platforms are available, and their utility depends upon factors such as the available technologies, number of data points required, budgets and the technical expertise required. Unfortunately, markers can rarely be exchanged between existing and newly developed platforms, meaning that previously generated data cannot be compared, or combined, with more recently generated data sets. We predict that genotyping by sequencing will become the predominant genotyping technology within the next 5–10 years. With this in mind, to ensure that data generated from current genotyping platforms continues to be of use, we have designed and utilized SNP ‐based capture probes from several thousand existing and publicly available probes from Axiom® and KASP ? genotyping platforms. We have validated our capture probes in a targeted genotyping by sequencing protocol using 31 previously genotyped UK elite hexaploid wheat accessions. Data comparisons between targeted genotyping by sequencing, Axiom® array genotyping and KASP ? genotyping assays, identified a set of 3256 probes which reliably bring together targeted genotyping by sequencing data with the previously available marker data set. As such, these probes are likely to be of considerable value to the wheat community. The probe details, full probe sequences and a custom built analysis pipeline may be freely downloaded from the CerealsDB website (http://www.cerealsdb.uk.net/cerealgenomics/CerealsDB /sequence_capture.php).     

Lesions Associated with Gastroduodenal Haemorrhage,in Relation to Aspirin Intake

The incidence of aspirin ingestion during the week preceding overt gastroduodenal bleeding was recorded in 582 patients. A positive aspirin history was found in 80% of patients with acute gastric lesions, in 63% of those in whom no lesion was found, in 52% of those with a chronic duodenal ulcer, and in 49% of patients with a chronic gastric ulcer. In a control series of 542 consecutive patients without overt bleeding admitted to the same wards during part of the time of this investigation the aspirin incidence was 32%.The difference in aspirin habits between these two series confirms that aspirin is a factor in precipitating overt haemorrhage in acute and chronic peptic ulcers, and that it is an important cause of bleeding from the stomach or duodenum, or both, in the absence of a chronic peptic ulcer.     

Ab initio molecular dynamics of the reactivity of vitamin C toward hydroxyl and $$ {HO}_2/{O}_2^{-} $$ radicals

Vitamin C is one of the most abundant exogenous antioxidants in the cell, and it is of the utmost importance to elucidate its mechanism of action against radicals. In this study, the reactivity of vitamin C toward OH and \( {HO}_2/{O}_2^{-} \) radicals in aqueous medium was analyzed by ab initio molecular dynamics using CPMD code. The simulations led to results similar to those of static studies or experiments for the pair of \( {HO}_2/{O}_2^{-} \) radicals but bring new insights for the reactivity with hydroxyl radical: the reaction takes place before the formation of an adduct and consists of two steps: first an electron is transferred to hydroxyl radical and then the ascorbyl radical loses a proton.

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Graphical Abstract Reactivity of vitamin C toward hydroxyl and \( {HO}_2/{O}_2^{-} \) radicals

     

Conformational analysis of macrocyclic frankincense (<Emphasis Type="Italic">Boswellia</Emphasis>) diterpenoids

Frankincense oleoresin has been used in traditional medicine for more than 5000 years. The phytochemistry of frankincense (Boswellia spp.) resins includes triterpenoids (including boswellic acids and their derivatives), diterpenoids (cembrenoids and cneorubenoids), and essential oils. The macrocyclic cembrene diterpenoids may play a part in the biological activities of frankincense resin, but neither the biological targets nor the modes of interaction with the targets are currently known. How these macrocycles interact with biological macromolecules likely depends on what conformation(s) are energetically available to them. In this work, a conformational analysis of 15 Boswellia cembrene diterpenoids and 1 verticillane diterpenoid was carried out at the B3LYP/6-31G* and M06-2X/6-31G* levels of theory, including the SM8 aqueous solvation model. The lowest-energy conformations of boscartin B and incensole oxide were the same as the previously reported X-ray crystal structures, while the lowest-energy conformations of boscartins A and C were very similar to the crystal structures. Boscartins D-H and isoincensole oxide showed only one low-energy conformation for each compound and are predicted to be conformationally locked. Incensole, isoincensolol, and serratol are predicted to be conformationally mobile with several low-energy forms. The conformational mobility of Boswellia cembrenoid diterpenoids depends largely on the degree of epoxidation, either oxirane or tetrahydrofuran rings.     

Evidence of multiple introductions and genetic admixture of the Asian brush-clawed shore crab <Emphasis Type="Italic">Hemigrapsus takanoi</Emphasis> (Decapoda: Brachyura: Varunidae) along the Northern European coast

The Asian brush-clawed shore crab Hemigrapsus takanoi is a non-indigenous species along the Northern European coast. Although the history of range expansion of European H. takanoi has been well-documented, little is known about the genetic compositions of either the introduced European populations or the native Asian ones. We therefore collected H. takanoi broadly from their native Asian sites and introduced European ranges, and genotyped them by sequencing the mitochondrial 16S RNA gene and by analyzing nuclear microsatellite loci. Our results revealed that the H. takanoi Bay of Seine (France) populations consisted of a genetic admixture between populations in Japan and those in the Yellow Sea region. These French populations should be carefully monitored in the future, since the genetic admixture of multiple source populations may accelerate range expansion in non-indigenous organisms. Our results also suggested that shipping lines from East Asia were more probable vectors than historical juvenile oyster transportations from Japan for the foundation of present European H. takanoi populations. Interestingly, gene flow between populations in Japan and those in the Yellow Sea region (i.e., domestic invasion) was not observed despite the higher potential for artificial translocations via shipping lines in the native Asian range compared with those from Asia to Europe. The lack of domestic invasions implied that intra-specific priority effects of the resident H. takanoi populations played an important role in preventing the successful colonization of artificially-transferred individuals.