Aggregates in blood filter chambers used from the plasma donations of anti-D donors: evaluation for monoclonal antibody discovery using phage display.

BACKGROUND: RhD-immunoglobulin (RhIg) prevents anti-D alloimmunisation in D-negative pregnant women when the fetus is D-positive, reducing the incidence of haemolytic disease of the fetus and newborn. Manufacturing RhIg is reliant on the limited supply of plasma donations with anti-D antibodies. Monoclonal antibody (mAb) development platforms such as phage display, require blood samples to be collected from anti-D donors, which may be a complicated process. The blood filter chamber (BFC) discarded after an anti-D donor's donation might provide a source of Ig-encoding RNA. This study aims to evaluate whether used BFCs are a suitable source of Ig-encoding RNA for phage display. MATERIAL AND METHODS: Haemonetics PCS2 BFCs were obtained from 10 anti-D donors for total RNA extraction, cDNA synthesis and amplification of VH and VL IgG sequences for assembly of single-chain variable fragments (scFvs). A scFv-phage display library was constructed and 3 rounds of biopanning were performed using D-positive and D-negative red blood cells (RBCs). Positive phage clones were isolated, Sanger sequenced and, where possible, reformatted into full-length human IgGs to define specificity. The BFC aggregates from 2 anti-D donors underwent a Wright-Giemsa stain and hematological cell count. RESULTS: Of 10 BFCs, a sufficient yield of total RNA for library construction was obtained from BFCs containing cellular aggregates (n=5). Aggregate analysis showed lymphocytes were the cellular source of Ig-encoding RNA. From the 5 samples with aggregates, scFvs were assembled from amplified IgG variable regions. The library constructed from 1 of these samples resulted in the isolation of clones binding to D-positive RBCs with IGHV3 gene usage. Of the 4 reformatted IgG, 3 were anti-D and 1 had undefined specificity. DISCUSSION: BFC aggregates are a new and convenient source of Ig-encoding RNA which can be used to construct Ig gene libraries for mAb isolation and discovery via antibody phage display

Assessing the Factorial Validity of the Attitudes and Belief Scale 2-Abbreviated Version: A Call for the Development a Gold Standard Method of Measuring Rational and Irrational Beliefs

Rational Emotive Behaviour Therapy (REBT) does not possess a measure of rational and irrational beliefs that meets internationally recognised standards for acceptable psychometric properties. Without such a measure the theory/practice of REBT cannot be rigorously evaluated, thus undermining its scientific veracity. The current study investigates the validity and reliability of a recently developed measure of rational and irrational beliefs: the Attitudes and Belief Scale 2-Abbreviated Version (ABS-2-AV). University students from three countries completed the ABS-2-AV (N = 397). An alternative models framework using confirmatory factor analysis indicated that a theoretically consistent eight-factor model of the ABS-2-AV provided the best fit of the data. A number of post-hoc modifications were required in order to achieve acceptable model fit results, and these modifications revealed important methodological limitations with the ABS-2-AV. Results indicated that the validity of the ABS-2-AV was undermined due to items measuring both the psychological process of interest (rational and irrational beliefs) and the context in which these beliefs processes are presented. This is a serious methodological limitation of the ABS-2 and all questionnaires derived from it, including the ABS-2-AV. This methodological limitation resulted in the ABS-2-AV possessing poor internal reliability. These limitations are discussed in relation to the broader REBT literature and the impact such problems have on research and practice. A call is made for REBT researchers to come together to develop a “gold standard” method of assessing rational and irrational beliefs that meets international standard for psychometric excellence

Dynamics of DNA replication loops reveal temporal control of lagging-strand synthesis

In all organisms, the protein machinery responsible for the replication of DNA, the replisome, is faced with a directionality problem. The antiparallel nature of duplex DNA permits the leading-strand polymerase to advance in a continuous fashion, but forces the lagging-strand polymerase to synthesize in the opposite direction. By extending RNA primers, the lagging-strand polymerase restarts at short intervals and produces Okazaki fragments. At least in prokaryotic systems, this directionality problem is solved by the formation of a loop in the lagging strand of the replication fork to reorient the lagging-strand DNA polymerase so that it advances in parallel with the leading-strand polymerase. The replication loop grows and shrinks during each cycle of Okazaki fragment synthesis. Here we use single-molecule techniques to visualize, in real time, the formation and release of replication loops by individual replisomes of bacteriophage T7 supporting coordinated DNA replication. Analysis of the distributions of loop sizes and lag times between loops reveals that initiation of primer synthesis and the completion of an Okazaki fragment each serve as a trigger for loop release. The presence of two triggers may represent a fail-safe mechanism ensuring the timely reset of the replisome after the synthesis of every Okazaki fragment.

Incommensurable worldviews? Is public use of complementary and alternative medicines incompatible with support for science and conventional medicine?

Proponents of controversial Complementary and Alternative Medicines, such as homeopathy, argue that these treatments can be used with great effect in addition to, and sometimes instead of, ?conventional? medicine. In doing so, they accept the idea that the scientific approach to the evaluation of treatment does not undermine use of and support for some of the more controversial CAM treatments. For those adhering to the scientific canon, however, such efficacy claims lack the requisite evidential basis from randomised controlled trials. It is not clear, however, whether such opposition characterises the views of the general public. In this paper we use data from the 2009 Wellcome Monitor survey to investigate public use of and beliefs about the efficacy of a prominent and controversial CAM within the United Kingdom, homeopathy. We proceed by using Latent Class Analysis to assess whether it is possible to identify a sub-group of the population who are at ease in combining support for science and conventional medicine with use of CAM treatments, and belief in the efficacy of homeopathy. Our results suggest that over 40% of the British public maintain positive evaluations of both homeopathy and conventional medicine simultaneously. Explanatory analyses reveal that simultaneous support for a controversial CAM treatment and conventional medicine is, in part, explained by a lack of scientific knowledge as well as concerns about the regulation of medical research

Globalisation and MA TESOL programs in the UK

This article reports the results of a mixed-methods approach to investigating the association between globalisation and MATESOL in UK universities. Qualitative and quantitative data collected from academic staff through eight emails, four interviews and 41 questionnaires indicate that the globalised context of higher education have affected these programmes in a number of ways including an increasing interest in recruiting more international students and a growing awareness about the need for curriculum and content modifications. The analysis of the data suggests that although change has been an inherent characteristic of these MAs over the past decade, it has been implemented gradually and conservatively, often relying on a dialectic relationship between academic staff and universities’ policies. The results imply that factors other than globalisation have also been at work. Many of the participants contend that globalisation has not lowered the quality of these MAs or standards of good practice

Is web interviewing a good alternative to telephone interviewing? Findings from the International Tobacco Control (ITC) Netherlands Survey

<p>Abstract</p> <p>Background</p> <p>Web interviewing is becoming increasingly popular worldwide, because it has several advantages over telephone interviewing such as lower costs and shorter fieldwork periods. However, there are also concerns about data quality of web surveys. The aim of this study was to compare the International Tobacco Control (ITC) Netherlands web and telephone samples on demographic and smoking related variables to assess differences in data quality.</p> <p>Methods</p> <p>Wave 1 of the ITC Netherlands Survey was completed by 1,668 web respondents and 404 telephone respondents of 18 years and older. The two surveys were conducted in parallel among adults who reported smoking at least monthly and had smoked at least 100 cigarettes over their lifetime.</p> <p>Results</p> <p>Both the web and telephone survey had a cooperation rate of 78%. Web respondents with a fixed line telephone were significantly more often married, had a lower educational level, and were older than web respondents without a fixed line telephone. Telephone respondents with internet access were significantly more often married, had a higher educational level, and were younger than telephone respondents without internet. Web respondents were significantly less often married and lower educated than the Dutch population of smokers. Telephone respondents were significantly less often married and higher educated than the Dutch population of smokers. Web respondents used the "don't know" options more often than telephone respondents. Telephone respondents were somewhat more negative about smoking, had less intention to quit smoking, and had more self efficacy for quitting. The known association between educational level and self efficacy was present only in the web survey.</p> <p>Conclusions</p> <p>Differences between the web and telephone sample were present, but the differences were small and not consistently favourable for either web or telephone interviewing. Our study findings suggested sometimes a better data quality in the web than in the telephone survey. Therefore, web interviewing can be a good alternative to telephone interviewing.</p

Does Proximity to Retailers Influence Alcohol and Tobacco Use Among Latino Adolescents?

Despite decades of research surrounding determinants of alcohol and tobacco (A&T) use among adolescents, built environment influences have only recently been explored. This study used ordinal regression on 205 Latino adolescents to explore the influence of the built environment (proximity to A&T retailers) on A&T use, while controlling for recognized social predictors. The sample was 45% foreign-born. A&T use was associated with distance from respondents’ home to the nearest A&T retailer (−), acculturation (+), parents’ consistent use of contingency management (−), peer use of A&T (+), skipping school (+), attending school in immediate proximity to the US/Mexico border (+), and the interaction between the distance to the nearest retailer and parents’ consistent use of contingency management (+). The association between decreasing distance to the nearest A&T retailer and increased A&T use in Latino adolescents reveals an additional risk behavior determinant in the US–Mexico border region

RNA-Seq Mapping and Detection of Gene Fusions with a Suffix Array Algorithm

High-throughput RNA sequencing enables quantification of transcripts (both known and novel), exon/exon junctions and fusions of exons from different genes. Discovery of gene fusions–particularly those expressed with low abundance– is a challenge with short- and medium-length sequencing reads. To address this challenge, we implemented an RNA-Seq mapping pipeline within the LifeScope software. We introduced new features including filter and junction mapping, annotation-aided pairing rescue and accurate mapping quality values. We combined this pipeline with a Suffix Array Spliced Read (SASR) aligner to detect chimeric transcripts. Performing paired-end RNA-Seq of the breast cancer cell line MCF-7 using the SOLiD system, we called 40 gene fusions among over 120,000 splicing junctions. We validated 36 of these 40 fusions with TaqMan assays, of which 25 were expressed in MCF-7 but not the Human Brain Reference. An intra-chromosomal gene fusion involving the estrogen receptor alpha gene ESR1, and another involving the RPS6KB1 (Ribosomal protein S6 kinase beta-1) were recurrently expressed in a number of breast tumor cell lines and a clinical tumor sample

Staphylococcus aureus Host Cell Invasion and Virulence in Sepsis Is Facilitated by the Multiple Repeats within FnBPA

Entry of Staphylococcus aureus into the bloodstream can lead to metastatic abscess formation and infective endocarditis. Crucial to the development of both these conditions is the interaction of S. aureus with endothelial cells. In vivo and in vitro studies have shown that the staphylococcal invasin FnBPA triggers bacterial invasion of endothelial cells via a process that involves fibronectin (Fn) bridging to α5β1 integrins. The Fn-binding region of FnBPA usually contains 11 non-identical repeats (FnBRs) with differing affinities for Fn, which facilitate the binding of multiple Fn molecules and may promote integrin clustering. We thus hypothesized that multiple repeats are necessary to trigger the invasion of endothelial cells by S. aureus. To test this we constructed variants of fnbA containing various combinations of FnBRs. In vitro assays revealed that endothelial cell invasion can be facilitated by a single high-affinity, but not low-affinity FnBR. Studies using a nisin-inducible system that controlled surface expression of FnBPA revealed that variants encoding fewer FnBRs required higher levels of surface expression to mediate invasion. High expression levels of FnBPA bearing a single low affinity FnBR bound Fn but did not invade, suggesting that FnBPA affinity for Fn is crucial for triggering internalization. In addition, multiple FnBRs increased the speed of internalization, as did higher expression levels of FnBPA, without altering the uptake mechanism. The relevance of these findings to pathogenesis was demonstrated using a murine sepsis model, which showed that multiple FnBRs were required for virulence. In conclusion, multiple FnBRs within FnBPA facilitate efficient Fn adhesion, trigger rapid bacterial uptake and are required for pathogenesis