Fragmentation of pooled PCR products for highly multiplexed TILLING

Improvements to massively parallel sequencing have allowed the routine recovery of natural and induced sequence variants. A broad range of biological disciplines have benefited from this, ranging from plant breeding to cancer research. The need for high sequence coverage to accurately recover single nucleotide variants and small insertions and deletions limits the applicability of whole genome approaches. This is especially true in organisms with a large genome size or for applications requiring the screening of thousands of individuals, such as the reverse-genetic technique known as TILLING. Using PCR to target and sequence chosen genomic regions provides an attractive alternative as the vast reduction in interrogated bases means that sample size can be dramatically increased through amplicon multiplexing and multidimensional sample pooling while maintaining suitable coverage for recovery of small mutations. Direct sequencing of PCR products is limited, however, due to limitations in read lengths of many next generation sequencers. In the present study we show the optimization and use of ultrasonication for the simultaneous fragmentation of multiplexed PCR amplicons for TILLING highly pooled samples. Sequencing performance was evaluated in a total of 32 pooled PCR products produced from 4096 chemically mutagenized Hordeum vulgare DNAs pooled in three dimensions. Evaluation of read coverage and base quality across amplicons suggests this approach is suitable for high-throughput TILLING and other applications employing highly pooled complex sampling schemes. Induced mutations previously identified in a traditional TILLING screen were recovered in this dataset further supporting the efficacy of the approach

Covered Cheatham-Platinum Stents for Aortic Coarctation Early and Intermediate-Term Results

ObjectivesThis study sought to evaluate the use of covered Cheatham-platinum (CP) stents in the treatment of aortic coarctation (CoA).BackgroundAortic aneurysms and stent fractures have been encountered after surgical and transcatheter treatment for CoA. Covered stents have previously been used in the treatment of abdominal and thoracic aneurysms in adults. We implanted covered CP stents as a rescue treatment in patients with CoA aneurysms or previous stent-related complications and in patients at risk of developing complications because of complex CoA anatomy or advanced age.MethodsThirty-three covered CP stents were implanted in 30 patients; 16 patients had had previous procedures. The remaining patients had complex or near-atretic CoA.ResultsThe mean patient age and weight were 28 (±17.5) years (range 8 to 65 years), and 62 (±13) kg (range 28 to 86 kg), respectively. The systolic gradient across the CoA decreased from a mean (±SD) of 36 ± 20 mm Hg before to a mean of 4 ± 4 mm Hg after the procedure (p < 0.0001), and the diameter of the CoA increased from 6.4 ± 3.8 mm to 17.1 ± 3.1 mm (p < 0.0001). The follow-up period was up to 40 months (mean, 11 months). All stents were patent and in good position on computed tomography or magnetic resonance imaging performed three to six months later. In 43% of the patients antihypertensive medication was either decreased or stopped.ConclusionsCovered CP stents may be used as the therapy of choice in patients with complications after CoA repairs, whereas they provide a safe alternative to conventional stenting in patients with severe and complex CoA lesions or advanced age

Efficacy and safety of the anti-IL-12/23 p40 monoclonal antibody, ustekinumab, in patients with active psoriatic arthritis despite conventional non-biological and biological anti-tumour necrosis factor therapy: 6-month and 1-year results of the phase 3, multicentre, double-blind, placebo-controlled, randomised PSUMMIT 2 trial

Objective: Assess ustekinumab efficacy (week 24/week 52) and safety (week 16/week 24/week 60) in patients with active psoriatic arthritis (PsA) despite treatment with conventional and/or biological anti-tumour necrosis factor (TNF) agents. Methods: In this phase 3, multicentre, placebo-controlled trial, 312 adults with active PsA were randomised (stratified by site, weight (&#8804;100 kg/&#62;100 kg), methotrexate use) to ustekinumab 45 mg or 90 mg at week 0, week 4, q12 weeks or placebo at week 0, week 4, week 16 and crossover to ustekinumab 45 mg at week 24, week 28 and week 40. At week 16, patients with &#60;5% improvement in tender/swollen joint counts entered blinded early escape (placebo→45 mg, 45 mg→90 mg, 90 mg→90 mg). The primary endpoint was &#8805;20% improvement in American College of Rheumatology (ACR20) criteria at week 24. Secondary endpoints included week 24 Health Assessment Questionnaire-Disability Index (HAQ-DI) improvement, ACR50, ACR70 and &#8805;75% improvement in Psoriasis Area and Severity Index (PASI75). Efficacy was assessed in all patients, anti-TNF-naïve (n=132) patients and anti-TNF-experienced (n=180) patients. Results: More ustekinumab-treated (43.8% combined) than placebo-treated (20.2%) patients achieved ACR20 at week 24 (p&#60;0.001). Significant treatment differences were observed for week 24 HAQ-DI improvement (p&#60;0.001), ACR50 (p&#8804;0.05) and PASI75 (p&#60;0.001); all benefits were sustained through week 52. Among patients previously treated with &#8805;1 TNF inhibitor, sustained ustekinumab efficacy was also observed (week 24 combined vs placebo: ACR20 35.6% vs 14.5%, PASI75 47.1% vs 2.0%, median HAQ-DI change −0.13 vs 0.0; week 52 ustekinumab-treated: ACR20 38.9%, PASI75 43.4%, median HAQ-DI change −0.13). No unexpected adverse events were observed through week 60. Conclusions: The interleukin-12/23 inhibitor ustekinumab (45/90 mg q12 weeks) yielded significant and sustained improvements in PsA signs/symptoms in a diverse population of patients with active PsA, including anti-TNF-experienced PsA patients

A highly mutagenised barley (cv. Golden Promise) TILLING population coupled with strategies for screening-by-sequencing

Background:We developed and characterised a highly mutagenised TILLING population of the barley (Hordeum vulgare) cultivar Golden Promise. Golden Promise is the 'reference' genotype for barley transformation and a primary objective of using this cultivar was to be able to genetically complement observed mutations directly in order to prove gene function. Importantly, a reference genome assembly of Golden Promise has also recently been developed. As our primary interest was to identify mutations in genes involved in meiosis and recombination, to characterise the population we focused on a set of 46 genes from the literature that are possible meiosis gene candidates. Results:Sequencing 20 plants from the population using whole exome capture revealed that the mutation density in this population is high (one mutation every 154 kb), and consequently even in this small number of plants we identified several interesting mutations. We also recorded some issues with seed availability and germination. We subsequently designed and applied a simple two-dimensional pooling strategy to identify mutations in varying numbers of specific target genes by Illumina short read pooled-amplicon sequencing and subsequent deconvolution. In parallel we assembled a collection of semi-sterile mutants from the population and used a custom exome capture array targeting the 46 candidate meiotic genes to identify potentially causal mutations. Conclusions:We developed a highly mutagenised barley TILLING population in the transformation competent cultivar Golden Promise. We used novel and cost-efficient screening approaches to successfully identify a broad range of potentially deleterious variants that were subsequently validated by Sanger sequencing. These resources combined with a high-quality genome reference sequence opens new possibilities for efficient functional gene validation.Miriam Schreiber, Abdellah Barakate, Nicola Uzrek, Malcolm Macaulay, Adeline Sourdille, Jenny Morris, Pete E. Hedley, Luke Ramsay and Robbie Waug

‘Death and his body-servant’: health, architecture and missionary endeavour at the Anelcauhat Mission House, Vanuatu

Remaining healthy was a major consideration for both indigenous and European peoples in the New Hebrides (now Vanuatu) during early contact. While local communities were often devastated by introduced disease, new missionaries sought practical ways to overcome the impact of tropical ailments that they considered to undermine the effectiveness of their activities. From the early 1850s onwards, Presbyterian missionaries in the southern New Hebrides began to construct ‘healthy’ homes, of which the surviving masonry mission house at Anelcauhat, Aneityum (1852-3) forms the earliest standing example. This paper draws on the results of both above- and in-ground archaeological recording to examine how the surviving structure reflects nineteenth-century ideas about illness and well-being before discussing the wider trajectory of such house construction, and associated matters connected with local communities, health and architecture that potentially impacted on missionary endeavour

Evaluation of nutritional information as an element influencing consumer behavior in gastronomy services

The aim of this study was the evaluation of nutritional information provided by frontline staff as an element influencing consumer behavior in gastronomy services. The empirical research was conducted under the project “The consumer in the market of catering services”, number 507-30-102-M00094-99, realized in the Chair of Gastronomy and Hospitality Management in the Faculty of Human Nutrition and Consumer Sciences at the Warsaw University of Life Sciences – SGGW. Quantitative research on the sample of 403 persons using purposive sampling were conducted in June 2016. The criteria of age (18–35), living in large cities as well as the fact of eating out. </jats:p

Prospects for the development of the sector of agricultural biogas plant in Poland

Produkcja i energetyczne wykorzystanie biogazu rolniczego jest jedną z metod pozyskiwania energii odnawialnej, która dopiero zaczyna rozwijać się w Polsce. Technologia ta jest natomiast rozpowszechniona i od wielu lat stosowana w krajach takich jak: Dania, Austria czy Niemcy. Polska posiada duży potencjał energetyczny krajowego rolnictwa. Zakłada on m.in. wykorzystanie produktów ubocznych rolnictwa oraz przemysłu rolno-spożywczego. Energetyczne wykorzystanie biogazu jest dla Polski szansą na spełnienie unijnych zobowiązań dotyczących produkcji energii z OZE. Obecnie inwestowanie w biogazownie rolnicze, wciąż wiąże się z dużą niepewnością, która wynika m.in. z braku transparentnego systemu wsparcia finansowego oraz licznych wahań w zakresie obowiązujących aktów prawnych.Energy production and use of agricultural biogas is one of methods of acquiring renewable energy, which is just beginning to develop in Poland. This technology is widespread and used for many years in countries such as Denmark, Austria or Germany. Poland has a large energy potential of the national agriculture. It assumes in using by-products of agriculture and agri-food industry. Energy use of biogas is a chance for Poland to fulfilment the EU's commitments on energy production from RES. Currently, investing in biogas plants, is still associated with large uncertainties that for ex. in the absence of a transparent system of financial support and numerous variations in terms of existing legislation