Quantitative Assessment of the Sensitivity of Various Commercial Reverse Transcriptases Based on Armored HIV RNA

The in-vitro reverse transcription of RNA to its complementary DNA, catalyzed by the enzyme reverse transcriptase, is the most fundamental step in the quantitative RNA detection in genomic studies. As such, this step should be as analytically sensitive, efficient and reproducible as possible, especially when dealing with degraded or low copy RNA samples. While there are many reverse transcriptases in the market, all claiming to be highly sensitive, there is need for a systematic independent comparison of their applicability in quantification of rare RNA transcripts or low copy RNA, such as those obtained from archival tissues.We performed RT-qPCR to assess the sensitivity and reproducibility of 11 commercially available reverse transcriptases in cDNA synthesis from low copy number RNA levels. As target RNA, we used a serially known number of Armored HIV RNA molecules, and observed that 9 enzymes we tested were consistently sensitive to ∼1,000 copies, seven of which were sensitive to ∼100 copies, while only 5 were sensitive to ∼10 RNA template copies across all replicates tested. Despite their demonstrated sensitivity, these five best performing enzymes (Accuscript, HIV-RT, M-MLV, Superscript III and Thermoscript) showed considerable variation in their reproducibility as well as their overall amplification efficiency. Accuscript and Superscript III were the most sensitive and consistent within runs, with Accuscript and Superscript II ranking as the most reproducible enzymes between assays.We therefore recommend the use of Accuscript or Superscript III when dealing with low copy number RNA levels, and suggest purification of the RT reactions prior to downstream applications (eg qPCR) to augment detection. Although the results presented in this study were based on a viral RNA surrogate, and applied to nucleic acid lysates derived from archival formalin-fixed paraffin embedded tissue, their relative performance on RNA obtained from other tissue types may vary, and needs future evaluation

A Novel Signaling Network Essential for Regulating Pseudomonas aeruginosa Biofilm Development

The important human pathogen Pseudomonas aeruginosa has been linked to numerous biofilm-related chronic infections. Here, we demonstrate that biofilm formation following the transition to the surface attached lifestyle is regulated by three previously undescribed two-component systems: BfiSR (PA4196-4197) harboring an RpoD-like domain, an OmpR-like BfmSR (PA4101-4102), and MifSR (PA5511-5512) belonging to the family of NtrC-like transcriptional regulators. These two-component systems become sequentially phosphorylated during biofilm formation. Inactivation of bfiS, bfmR, and mifR arrested biofilm formation at the transition to the irreversible attachment, maturation-1 and -2 stages, respectively, as indicated by analyses of biofilm architecture, and protein and phosphoprotein patterns. Moreover, discontinuation of bfiS, bfmR, and mifR expression in established biofilms resulted in the collapse of biofilms to an earlier developmental stage, indicating a requirement for these regulatory systems for the development and maintenance of normal biofilm architecture. Interestingly, inactivation did not affect planktonic growth, motility, polysaccharide production, or initial attachment. Further, we demonstrate the interdependency of this two-component systems network with GacS (PA0928), which was found to play a dual role in biofilm formation. This work describes a novel signal transduction network regulating committed biofilm developmental steps following attachment, in which phosphorelays and two sigma factor-dependent response regulators appear to be key components of the regulatory machinery that coordinates gene expression during P. aeruginosa biofilm development in response to environmental cues

Migraine day frequency in migraine prevention: longitudinal modelling approaches

Background Health economic models are critical tools to inform reimbursement agencies on health care interventions. Many clinical trials report outcomes using the frequency of an event over a set period of time, for example, the primary efficacy outcome in most clinical trials of migraine prevention is mean change in the frequency of migraine days (MDs) per 28 days (monthly MDs [MMD]) relative to baseline for active treatment versus placebo. Using these cohort-level endpoints in economic models, accounting for variation among patients is challenging. In this analysis, parametric models of change in MMD for migraine preventives were assessed using data from erenumab clinical studies. Methods MMD observations from the double-blind phases of two studies of erenumab were used: one in episodic migraine (EM) (NCT02456740) and one in chronic migraine (CM) (NCT02066415). For each trial, two longitudinal regression models were fitted: negative binomial and beta binomial. For a thorough comparison we also present the fitting from the standard multilevel Poisson and the zero inflated negative binomial. Results Using the erenumab study data, both the negative binomial and beta-binomial models provided unbiased estimates relative to observed trial data with well-fitting distribution at various time points. Conclusions This proposed methodology, which has not been previously applied in migraine, has shown that these models may be suitable for estimating MMD frequency. Modelling MMD using negative binomial and beta-binomial distributions can be advantageous because these models can capture intra- and inter-patient variability so that trial observations can be modelled parametrically for the purposes of economic evaluation of migraine prevention. Such models have implications for use in a wide range of disease areas when assessing repeated measured utility values

The formation of precursor structures in cylindrical and "4 x 4" wire arrays

This paper summarizes observations of precursor-column formation in cylindrical wire arrays on the 1-MA MAGPIE generator at Imperial College London. Direct experimental measurements of the diameter variation during the collapse and formation phase of the precursor column taken from gated soft X-ray images are presented. Three stages in precursor-column formation are identifiable from the data: broad initial density profile, rapid contraction to small diameter, and slow expansion after formation. The variation in the minimum diameter is measured for several materials, and data show good agreement with a pressure-balance model. Stability calculations suggest that the fraction of the drive current flowing the precursor column is < 1% for Al arrays and similar to 2% for W. "4 x 4" arrays show the formation of local precursor columns close to the wire position, which exhibit differences in stability depending on the wire separation. This may be related to deceleration of the plasma flow by the B-field or current convection in the flow

Benchmarking of trauma care worldwide: the potential value of an International Trauma Data Bank (ITDB)

Background: National trauma registries have helped improve patient outcomes across the world. Recently, the idea of an International Trauma Data Bank (ITDB) has been suggested to establish global comparative assessments of trauma outcomes. The objective of this study was to determine whether global trauma data could be combined to perform international outcomes benchmarkingMethods: We used observed/expected (O/E) mortality ratios to compare two trauma centers [European high-income country (HIC) and Asian lower-middle income country (LMIC)] with centers in the North American National Trauma Data Bank (NTDB). Patients (≥16 years) with blunt/penetrating injuries were included. Multivariable logistic regression, adjusting for known predictors of trauma mortality, was performed. Estimates were used to predict the expected deaths at each center and to calculate O/E mortality ratios for benchmarking. Results: A total of 375,433 patients from 301 centers were included from the NTDB (2002–2010). The LMIC trauma center had 806 patients (2002–2010), whereas the HIC reported 1,003 patients (2002–2004). The most important known predictors of trauma mortality were adequately recorded in all datasets. Mortality benchmarking revealed that the HIC center performed similarly to the NTDB centers [O/E = 1.11 (95 % confidence interval (CI) 0.92–1.35)], whereas the LMIC center showed significantly worse survival [O/E = 1.52 (1.23–1.88)]. Subset analyses of patients with blunt or penetrating injury showed similar results.Conclusions: Using only a few key covariates, aggregated global trauma data can be used to adequately perform international trauma center benchmarking. The creation of the ITDB is feasible and recommended as it may be a pivotal step towards improving global trauma outcomes. This work was declared as the best oral presentation at the International Association for Trauma Surgery and Intensive Care/the American Association for the Surgery of Trauma (IATSIC/AAST) Scientific Paper Session at the International Surgical Week (ISW) 2013, Helsinki, Finland