pcrEfficiency: a Web tool for PCR amplification efficiency prediction

<p>Abstract</p> <p>Background</p> <p>Relative calculation of differential gene expression in quantitative PCR reactions requires comparison between amplification experiments that include reference genes and genes under study. Ignoring the differences between their efficiencies may lead to miscalculation of gene expression even with the same starting amount of template. Although there are several tools performing PCR primer design, there is no tool available that predicts PCR efficiency for a given amplicon and primer pair.</p> <p>Results</p> <p>We have used a statistical approach based on 90 primer pair combinations amplifying templates from bacteria, yeast, plants and humans, ranging in size between 74 and 907 bp to identify the parameters that affect PCR efficiency. We developed a generalized additive model fitting the data and constructed an open source Web interface that allows the obtention of oligonucleotides optimized for PCR with predicted amplification efficiencies starting from a given sequence.</p> <p>Conclusions</p> <p>pcrEfficiency provides an easy-to-use web interface allowing the prediction of PCR efficiencies prior to web lab experiments thus easing quantitative real-time PCR set-up. A web-based service as well the source code are provided freely at <url>http://srvgen.upct.es/efficiency.html</url> under the GPL v2 license.</p

The diagnostic value of endoscopy and Helicobacter pylori tests for peptic ulcer patients in late post-treatment setting

BACKGROUND: Guidelines for management of peptic ulcer patients after the treatment are largely directed to detection of H. pylori infection using only non-invasive tests. We compared the diagnostic value of non-invasive and endoscopy based H. pylori tests in a late post-treatment setting. METHODS: Altogether 34 patients with dyspeptic complaints were referred for gastroscopy 5 years after the treatment of peptic ulcer using a one-week triple therapy scheme. The endoscopic and histologic findings were evaluated according to the Sydney classification. Bacteriological, PCR and cytological investigations and (13)C-UBT tests were performed. RESULTS: Seventeen patients were defined H. pylori positive by (13)C-UBT test, PCR and histological examination. On endoscopy, peptic ulcer persisted in 4 H. pylori positive cases. Among the 6 cases with erosions of the gastric mucosa, only two patients were H. pylori positive. Mucosal atrophy and intestinal metaplasia were revealed both in the H. pylori positive and H. pylori negative cases. Bacteriological examination revealed three clarithromycin resistant H. pylori strains. Cytology failed to prove validity for diagnosing H. pylori in a post-treatment setting. CONCLUSIONS: In a late post-treatment setting, patients with dyspepsia should not be monitored only by non-invasive investigation methods; it is also justified to use the classical histological evaluation of H. pylori colonisation, PCR and bacteriology as they have shown good concordance with (13)C-UBT. Moreover, endoscopy and histological investigation of a gastric biopsy have proved to be the methods with an additional diagnostic value, providing the physician with information about inflammatory, atrophic and metaplastic lesions of the stomach in dyspeptic H. pylori positive and negative patients. Bacteriological methods are suggested for detecting the putative antimicrobial resistance of H. pylori, aimed at successful eradication of infection in persistent peptic ulcer cases

Missed, not missing: Phylogenomic evidence for the existence of Avian FoxP3

The Forkhead box transcription factor FoxP3 is pivotal to the development and function of regulatory T cells (Tregs), which make a major contribution to peripheral tolerance. FoxP3 is believed to perform a regulatory role in all the vertebrate species in which it has been detected. The prevailing view is that FoxP3 is absent in birds and that avian Tregs rely on alternative developmental and suppressive pathways. Prompted by the automated annotation of foxp3 in the ground tit (Parus humilis) genome, we have questioned this assumption. Our analysis of all available avian genomes has revealed that the foxp3 locus is missing, incomplete or of poor quality in the relevant genomic assemblies for nearly all avian species. Nevertheless, in two species, the peregrine falcon (Falco peregrinus) and the saker falcon (F. cherrug), there is compelling evidence for the existence of exons showing synteny with foxp3 in the ground tit. A broader phylogenomic analysis has shown that FoxP3 sequences from these three species are similar to crocodilian sequences, the closest living relatives of birds. In both birds and crocodilians, we have also identified a highly proline-enriched region at the N terminus of FoxP3, a region previously identified only in mammals

Rationale, study design, and analysis plan of the Alveolar Recruitment for ARDS Trial (ART): Study protocol for a randomized controlled trial

Background: Acute respiratory distress syndrome (ARDS) is associated with high in-hospital mortality. Alveolar recruitment followed by ventilation at optimal titrated PEEP may reduce ventilator-induced lung injury and improve oxygenation in patients with ARDS, but the effects on mortality and other clinical outcomes remain unknown. This article reports the rationale, study design, and analysis plan of the Alveolar Recruitment for ARDS Trial (ART). Methods/Design: ART is a pragmatic, multicenter, randomized (concealed), controlled trial, which aims to determine if maximum stepwise alveolar recruitment associated with PEEP titration is able to increase 28-day survival in patients with ARDS compared to conventional treatment (ARDSNet strategy). We will enroll adult patients with ARDS of less than 72 h duration. The intervention group will receive an alveolar recruitment maneuver, with stepwise increases of PEEP achieving 45 cmH(2)O and peak pressure of 60 cmH2O, followed by ventilation with optimal PEEP titrated according to the static compliance of the respiratory system. In the control group, mechanical ventilation will follow a conventional protocol (ARDSNet). In both groups, we will use controlled volume mode with low tidal volumes (4 to 6 mL/kg of predicted body weight) and targeting plateau pressure &lt;= 30 cmH2O. The primary outcome is 28-day survival, and the secondary outcomes are: length of ICU stay; length of hospital stay; pneumothorax requiring chest tube during first 7 days; barotrauma during first 7 days; mechanical ventilation-free days from days 1 to 28; ICU, in-hospital, and 6-month survival. ART is an event-guided trial planned to last until 520 events (deaths within 28 days) are observed. These events allow detection of a hazard ratio of 0.75, with 90% power and two-tailed type I error of 5%. All analysis will follow the intention-to-treat principle. Discussion: If the ART strategy with maximum recruitment and PEEP titration improves 28-day survival, this will represent a notable advance to the care of ARDS patients. Conversely, if the ART strategy is similar or inferior to the current evidence-based strategy (ARDSNet), this should also change current practice as many institutions routinely employ recruitment maneuvers and set PEEP levels according to some titration method.Hospital do Coracao (HCor) as part of the Program 'Hospitais de Excelencia a Servico do SUS (PROADI-SUS)'Brazilian Ministry of Healt