Isolation of polymorphic microsatellites in the stemless thistle (Cirsium acaule) and their utility in other Cirsium species

The genus Cirsium includes species with both widespread and restricted geographical distributions, several of which are serious weeds. Nine polymorphic microsatellite loci were isolated from the stemless thistle Cirsium acaule. Eight were polymorphic in C. acaule, six in C. arvense and seven in C. heterophyllum. One locus monomorphic in C. acaule showed polymorphism in C. heterophyllum. The mean number of alleles per locus was 4.1 in C. acaule, 6.2 in C. arvense and 2.9 in C. heterophyllum. These nine loci were also amplified in C. eriophorum and C. vulgare, suggesting that these markers may be of use throughout the genus

A post-labeling method for multiplexed and multicolored genotyping analysis of SSR, indel and SNP markers in single tube with bar-coded split tag (BStag)

<p>Abstract</p> <p>Background</p> <p>Genotyping analysis using capillary DNA sequencing with fluorescently labeled primer pairs obtained by polymerase chain reaction (PCR) is widely used, but is expensive. The post-PCR labeling method using fluorescently labeled short oligonucleotides and nested PCR of the amplified product obtained from unlabeled primer pairs is a simple and inexpensive alternative. However, previously reported protocols often produced spurious peaks or inconsistent amplification under multiplexed analysis as a result of simultaneous progress of both the amplification and labeling reactions and local homology of the attached tag sequence.</p> <p>Results</p> <p>A set of 16 bp-long oligonucleotide sequences termed bar-coded split tag (BStag), comprising a common basal region, a three-nucleotide 'bar-code' sequence, and a mismatched nucleotide at the middle position were designed for selective post-PCR labeling. The BStag was attached at the 5' end of the forward primer of interest. The melting temperature of the BStag was low enough to separate the labeling reaction from initial PCR amplification, and each sequence was minimally divergent but maintained maximum selectivity. Post-PCR labeling of the amplified product was achieved by extending for three cycles at a lower annealing temperature after the conventional amplification program with the appropriate fluorescently labeled BStag primer. No amplification was confirmed with BStag primers for 12 plant species. The electropherogram of the labeled product obtained using this method was consistent with that of prelabeled primer, except for their apparent size.</p> <p>Conclusions</p> <p>BStag enabled multiplexed post-PCR labeling of simple sequence repeat or insertion/deletion markers with different dyes in a single tube. BStag in conjunction with locus specific oligo and allele specific oligo was also useful for single nucleotide polymorphism analysis. The labeling protocol was simple and no additional operation was required. Single-tube multiplexed post-PCR labeling is useful for a wide variety of genotyping studies with maximal flexibility and minimal costs.</p

Experimental evidence that livestock grazing intensity affects cyclic vole population regulation processes

Peer reviewedPublisher PD

Variation of 52 new Y-STR loci in the Y Chromosome Consortium worldwide panel of 76 diverse individuals

We have established 16 small multiplex reactions of two–four loci to amplify 52 recently described single-copy simple Y-STRs and typed these loci in a worldwide panel of 74 diverse men and two women. Two Y-STRs were found to be commonly multicopy in this sample set and were excluded from the study. Of the remaining 50, four (DYS481, DYS570, DYS576 and DYS643) showed higher diversities than the commonly used loci and can potentially provide increased haplotype discrimination in both forensic and anthropological work. Ten loci showed occasional missing alleles, duplicated peaks or intermediate-sized alleles. ELECTRONIC SUPPLEMENTARY MATERIAL: Supplementary material is available for this article at http://dx.doi.org/10.1007/s00414-006-0124-8 and is accessible for authorized users

Enhancement drugs: are there limits to what we should enhance and why?

Substances, such as alcohol, opiates and cannabis, have been used by humans for millennia. Today, a much wider range of substances are used for a range of purposes, including the enhancement of performance during university studies, sexual experiences, sports, exercise, at celebrations, socializing and the experience of art and music. Substance use is also associated with a range of harmful effects to the individual and society as a whole. Prohibitions, regulation, prevention and treatment have all been used to protect against this harm. In this commentary, it is argued that public health interventions should target relevant harms and not to evaluate which aspects of human endeavors and experiences should be enhanced and which should not. It is argued that interventions should directly target the harmful effects, using the best available evidence. Two examples are given of substances that may be altered to prevent serious harm - one for alcohol and one for cannabis. In the case of alcohol, the addition of dissolved oxygen could reduce both the risk of accidents and the risk of liver damage associated with alcohol consumption. In the case of cannabis, there is strong indication that the reduction of content Δ-tetrahydrocannabinol and the increase of cannabidiol could reduce the risk of psychoses and the addiction associated with its use. The aim of this article is to show that responsible regulation should not necessarily be restricted to preventing the use and/or (in the case of alcohol) a reduction in the amounts and frequency of its use, but should also aim to include a range of other strategies that could reduce the burden of illness associated with illicit substance use

Deletion of PTH Rescues Skeletal Abnormalities and High Osteopontin Levels in Klotho−/− Mice

Maintenance of normal mineral ion homeostasis is crucial for many biological activities, including proper mineralization of the skeleton. Parathyroid hormone (PTH), Klotho, and FGF23 have been shown to act as key regulators of serum calcium and phosphate homeostasis through a complex feedback mechanism. The phenotypes of Fgf23−/− and Klotho−/− (Kl−/−) mice are very similar and include hypercalcemia, hyperphosphatemia, hypervitaminosis D, suppressed PTH levels, and severe osteomalacia/osteoidosis. We recently reported that complete ablation of PTH from Fgf23−/− mice ameliorated the phenotype in Fgf23−/−/PTH−/− mice by suppressing serum vitamin D and calcium levels. The severe osteomalacia in Fgf23−/− mice, however, persisted, suggesting that a different mechanism is responsible for this mineralization defect. In the current study, we demonstrate that deletion of PTH from Kl−/− (Kl−/−/PTH−/− or DKO) mice corrects the abnormal skeletal phenotype. Bone turnover markers are restored to wild-type levels; and, more importantly, the skeletal mineralization defect is completely rescued in Kl−/−/PTH−/− mice. Interestingly, the correction of the osteomalacia is accompanied by a reduction in the high levels of osteopontin (Opn) in bone and serum. Such a reduction in Opn levels could not be observed in Fgf23−/−/PTH−/− mice, and these mice showed sustained osteomalacia. This significant in vivo finding is corroborated by in vitro studies using calvarial osteoblast cultures that show normalized Opn expression and rescued mineralization in Kl−/−/PTH−/− mice. Moreover, continuous PTH infusion of Kl−/− mice significantly increased Opn levels and osteoid volume, and decreased trabecular bone volume. In summary, our results demonstrate for the first time that PTH directly impacts the mineralization disorders and skeletal deformities of Kl−/−, but not of Fgf23−/− mice, possibly by regulating Opn expression. These are significant new perceptions into the role of PTH in skeletal and disease processes and suggest FGF23-independent interactions of PTH with Klotho

Optimization and validation of multi-coloured capillary electrophoresis for genotyping of Plasmodium falciparum merozoite surface proteins (msp1 and 2)

BACKGROUND: Genotyping of Plasmodium falciparum based on PCR amplification of the polymorphic genes encoding the merozoite surface proteins 1 and 2 (msp1 and msp2) is well established in the field of malaria research to determine the number and types of concurrent clones in an infection. Genotyping is regarded essential in anti-malarial drug trials to define treatment outcome, by distinguishing recrudescent parasites from new infections. Because of the limitations in specificity and resolution of gel electrophoresis used for fragment analysis in most genotyping assays it became necessary to improve the methodology. An alternative technique for fragment analysis is capillary electrophoresis (CE) performed using automated DNA sequencers. Here, one of the most widely-used protocols for genotyping of P. falciparum msp1 and msp2 has been adapted to the CE technique. The protocol and optimization process as well as the potentials and limitations of the technique in molecular epidemiology studies and anti-malarial drug trials are reported. METHODS: The original genotyping assay was adapted by fluorescent labeling of the msp1 and msp2 allelic type specific primers in the nested PCR and analysis of the final PCR products in a DNA sequencer. A substantial optimization of the fluorescent assay was performed. The CE method was validated using known mixtures of laboratory lines and field samples from Ghana and Tanzania, and compared to the original PCR assay with gel electrophoresis. RESULTS: The CE-based method showed high precision and reproducibility in determining fragment size (< 1 bp). More genotypes were detected in mixtures of laboratory lines and blood samples from malaria infected children, compared to gel electrophoresis. The capacity to distinguish recrudescent parasites from new infections in an anti-malarial drug trial was similar by both methods, resulting in the same outcome classification, however with more precise determination by CE. CONCLUSION: The improved resolution and reproducibility of CE in fragment sizing allows for comparison of alleles between separate runs and determination of allele frequencies in a population. The more detailed characterization of individual msp1 and msp2 genotypes may contribute to improved assessments in anti-malarial drug trials and to a further understanding of the molecular epidemiology of these polymorphic P. falciparum antigens