Ice-COLD-PCR enables rapid amplification and robust enrichment for low-abundance unknown DNA mutations

Identifying low-abundance mutations within wild-type DNA is important in several fields of medicine, including cancer, prenatal diagnosis and infectious diseases. However, utilizing the clinical and diagnostic potential of rare mutations is limited by sensitivity of the molecular techniques employed, especially when the type and position of mutations are unknown. We have developed a novel platform that incorporates a synthetic reference sequence within a polymerase chain reaction (PCR) reaction, designed to enhance amplification of unknown mutant sequences during COLD-PCR (CO-amplification at Lower Denaturation temperature). This new platform enables an Improved and Complete Enrichment (ice-COLD-PCR) for all mutation types and eliminates shortcomings of previous formats of COLD-PCR. We evaluated ice-COLD-PCR enrichment in regions of TP53 in serially diluted mutant and wild-type DNA mixtures. Conventional-PCR, COLD-PCR and ice-COLD-PCR amplicons were run in parallel and sequenced to determine final mutation abundance for a range of mutations representing all possible single base changes. Amplification by ice-COLD-PCR enriched all mutation types and allowed identification of mutation abundances down to 1%, and 0.1% by Sanger sequencing or pyrosequencing, respectively, surpassing the capabilities of other forms of PCR. Ice-COLD-PCR will help elucidate the clinical significance of low-abundance mutations and our understanding of cancer origin, evolution, recurrence-risk and treatment diagnostics

A numerical investigation of moment coefficient and flow structure in a rotor-stator cavity with rotor mounted bolts

This paper presents a numerical study of the effect of rotor mounted bolts on the moment coefficient and flow structure within a rotor-stator cavity representative of modern gas turbine engine design. The CFD computations are performed using the commercial code FLUENT. The simulation methodology is first validated using experimental data from plain co-rotating disc and rotor-stator cavities from the open literature. Comparisons are then made with experimental data obtained from a test rig at the Thermo Fluid Mechanics Research Centre (TFMRC), University of Sussex. Computations were performed at Reφ = 6.8 × 106, Cw = 5929 (λT = 0.35) with different numbers of bolts (0 < N < 60), and also a continuous ring, at r/b = 0.9. The study has improved the current understanding of the effect on moment coefficient and flow structure that rotor mounted protrusions have in rotor-stator systems. It is seen that the contribution of skin friction to the moment coefficient reduces as the number of bolts is increased. The size and shape of the wake created by a rotating bolt also means that the pressure loss per bolt reduces with N but the overall effect is to increase the moment coefficient because there are more bolts. Copyright © 2011 by ASME

Engagement-related process factors in services for street-connected children and young people in low and middle income countries: a thematic synthesis

A recent Cochrane/ Campbell systematic review of interventions for promoting reintegration and reducing harmful behaviour and lifestyles in street-connected children and young people, supported by the International Initiative for Impact Evaluation (3ie), identified 11 studies evaluating 12 different interventions which met the inclusion criteria (Coren et al. 2013). The research studies had to contain a comparison group and look at street-connected children and young people between the ages of 0 and 24. None of the included studies were from low- and middle-income countries (LMICs), or involved process evaluations. The current thematic synthesis focused on engagement processes and strategies in studies pertaining to LMICs but excluded from the review on methodological grounds, complemented by studies identified through a search update conducted in March 2013. We included twenty-seven qualitative or mixed methods studies, which examined broad range of interventions and services adopted in 21 LMICs. The synthesis methods were primarily qualitative. On the basis of our findings, we conceptualised engagement as consisting of outreach and sustaining phases. Three separate dimensions emerged in the sustaining phases: meeting multiple needs, relations to adult service users, and community and family engagement. Our synthesis outlines challenges and dilemmas of engagement in each of these areas. The data also highlighted some common shortcomings in the service sector in LMICs, including lack of appropriate professional training and service development, lack of funding, and lack of co-operation with other relevant agencies. Innovative approaches to engagement, such as participatory initiatives and partnerships with universities, are also discussed

Three dimensional rotational angiography imaging of double aortic arch vascular ring

Three dimensional (3D) rotational angiography is a technique used increasingly for imaging in congenital heart disease. Here the use of this technique for imaging of double aortic arch vascular ring is described and the advantages of this modality. are discussed. 3D rotational angiography is an excellent tool for imaging of various vascular anomalies. It provides high quality accurate images through a quick and safe procedure.peer-reviewe

Assortative human pair-bonding for partner ancestry and allelic variation of the dopamine receptor D4 (DRD4) gene

The 7R allele of the dopamine receptor D4 gene has been associated with attention-deficit hyperactivity disorder and risk taking. On the cross-population scale, 7R allele frequencies have been shown to be higher in populations with more of a history of long-term migrations. It has also been shown that the 7R allele is associated with individuals having multiple-ancestries. Here we conduct a replication of this latter finding with two independent samples. Measures of subjects&#x2019; ancestry are used to examine past reproductive bonds. The individuals&#x2019; history of inter-racial/ancestral dating and their feelings about this are also assessed. Tentative support for an association between multiple ancestries and the 7R allele were found. These results are dependent upon the method of questioning subjects about their ancestries. Inter-racial dating and feelings about inter-racial pairing were not related to the presence of the 7R allele. This might be accounted for by secular trends that might have substantively altered the decision-making process employed when considering relationships with individuals from different groups. This study provides continued support for the 7R allele playing a role in migration and/or mate choice patterns. However, replications and extensions of this study are needed and must carefully consider how ancestry/race is assessed

Fragmentation of the large subunit ribosomal RNA gene in oyster mitochondrial genomes

<p>Abstract</p> <p>Background</p> <p>Discontinuous genes have been observed in bacteria, archaea, and eukaryotic nuclei, mitochondria and chloroplasts. Gene discontinuity occurs in multiple forms: the two most frequent forms result from introns that are spliced out of the RNA and the resulting exons are spliced together to form a single transcript, and fragmented gene transcripts that are not covalently attached post-transcriptionally. Within the past few years, fragmented ribosomal RNA (rRNA) genes have been discovered in bilateral metazoan mitochondria, all within a group of related oysters.</p> <p>Results</p> <p>In this study, we have characterized this fragmentation with comparative analysis and experimentation. We present secondary structures, modeled using comparative sequence analysis of the discontinuous mitochondrial large subunit rRNA genes of the cupped oysters <it>C. virginica, C. gigas</it>, and <it>C. hongkongensis</it>. Comparative structure models for the large subunit rRNA in each of the three oyster species are generally similar to those for other bilateral metazoans. We also used RT-PCR and analyzed ESTs to determine if the two fragmented LSU rRNAs are spliced together. The two segments are transcribed separately, and not spliced together although they still form functional rRNAs and ribosomes.</p> <p>Conclusions</p> <p>Although many examples of discontinuous ribosomal genes have been documented in bacteria and archaea, as well as the nuclei, chloroplasts, and mitochondria of eukaryotes, oysters are some of the first characterized examples of fragmented bilateral animal mitochondrial rRNA genes. The secondary structures of the oyster LSU rRNA fragments have been predicted on the basis of previous comparative metazoan mitochondrial LSU rRNA structure models.</p

COLD-PCR Amplification of Bisulfite-Converted DNA Allows the Enrichment and Sequencing of Rare Un-Methylated Genomic Regions

Aberrant hypo-methylation of DNA is evident in a range of human diseases including cancer and diabetes. Development of sensitive assays capable of detecting traces of un-methylated DNA within methylated samples can be useful in several situations. Here we describe a new approach, fast-COLD-MS-PCR, which amplifies preferentially un-methylated DNA sequences. By employing an appropriate denaturation temperature during PCR of bi-sulfite converted DNA, fast-COLD-MS-PCR enriches un-methylated DNA and enables differential melting analysis or bisulfite sequencing. Using methylation on the MGMT gene promoter as a model, it is shown that serial dilutions of controlled methylation samples lead to the reliable sequencing of un-methylated sequences down to 0.05% un-methylated-to-methylated DNA. Screening of clinical glioma tumor and infant blood samples demonstrated that the degree of enrichment of un-methylated over methylated DNA can be modulated by the choice of denaturation temperature, providing a convenient method for analysis of partially methylated DNA or for revealing and sequencing traces of un-methylated DNA. Fast-COLD-MS-PCR can be useful for the detection of loss of methylation/imprinting in cancer, diabetes or diet-related methylation changes

PROTOCOL: The effectiveness of psychosocial interventions for reducing problematic substance use, improving mental health, and improving housing stability for adults experiencing homelessness: A systematic review and meta-analysis

This systematic review is part of a broader evidence synthesis which aims to produce two systematic reviews to address a significant gap in the evidence base identified by Luchenski et al. (2018) and by (White, 2018). The first review (which is the subject of this protocol) will use meta-analysis to examine the effectiveness of different psychosocial interventions in (1) reducing problematic substance use; (2) improving mental health; and (3) improving housing stability for adults experiencing homelessness. The second review (which is covered by a separate title registration and protocol) will be of the experiences of adults experiencing homelessness when accessing or using psychosocial interventions, and will be a qualitative evidence synthesis using thematic synthesis (Thomas & Harden, 2008)

Supporting ethical, independent learning behavior among university students in the Arabian Gulf

© Springer International Publishing Switzerland 2014. Students in the Arabian Gulf region and the world over confront plagiarism temptations wittingly or unwittingly due to the multitude of free and easily available electronic sources of information. Rather than develop independent learning skills and academic integrity, students are often taken advantage of by essay mills that sell readymade essays. Instructors at times compound the problem by repeatedly recycling course assignments and tasks. Furthermore, there have been reports of the use of social networking sites for outsourcing and contract cheating in student assignments. This paper discusses how educators in an institution of higher learning in the UAE assist students to develop good academic skills. Data collection is with the use of an online survey questionnaire. Concurring with Wheeler and Anderson (2010) who call for appropriate and comprehensive institutional policies and guidelines for dealing with plagiarism, practical examples of the processes and procedures used at these institutions are provided