Development of a single tube 640-plex genotyping method for detection of nucleic acid variations on microarrays

Detection of DNA sequence variation is critical to biomedical applications, including disease genetic identification, diagnosis and treatment, drug discovery and forensic analysis. Here, we describe an arrayed primer extension-based genotyping method (APEX-2) that allows multiplex (640-plex) DNA amplification and detection of single nucleotide polymorphisms (SNPs) and mutations on microarrays via four-color single-base primer extension. The founding principle of APEX-2 multiplex PCR requires two oligonucleotides per SNP/mutation to generate amplicons containing the position of interest. The same oligonucleotides are then subsequently used as immobilized single-base extension primers on a microarray. The method described here is ideal for SNP or mutation detection analysis, molecular diagnostics and forensic analysis. This robust genetic test has minimal requirements: two primers, two spots on the microarray and a low cost four-color detection system for the targeted site; and provides an advantageous alternative to high-density platforms and low-density detection systems

Procjena izloženosti UV zračenju tijekom ljetnih mjeseci u Hrvatskoj s pomoću jednostavne približne formule

The Tropospheric Ultraviolet-Visible (TUV) model, version 4.2 developed by Madronich (2003) was usedto estimate the extent of ultraviolet (UV) exposure of general population in Croatia over the summer. Solarnoon values (13 h local time, CEST) of the ultraviolet index (UVI) for the period April to October 2004 were calculated for 61 cities in Croatia. The results showed that the risk of sunburn at 13 h local time inclear weather was high between April and September (UVI >7) and very high in July (UVI >10). In July, the UVI exceeded 8 between 11 h and 15 h local time. In this study, we developed a simple approximate formula to estimate UVI. The formula includes data on the time, date, altitude and clouds. The difference between our estimate and the TUV model for the summer months of June, July and August at 10 h to16 h local time was less than 10 %.Tropospheric Ultraviolet-Visible (TUV) model, verzija 4.2 autora S. Madronicha (2003.) upotrijebljen je zaprocjenu izloženosti ultraljubičastom (UV) zračenju stanovništva u Hrvatskoj. Podnevne vrijednosti (13 hprema lokalnom vremenu) ultraljubičastog indeksa (UVI) izračunane su za 61 mjesto u Hrvatskoj za razdobljetravanj - listopad. Rezultati pokazuju da je u 13 h prema lokalnom vremenu rizik od nastanka opeklina izazvanih sunčevim zračenjem u danima bez naoblake visok između travnja i rujna (UVI > 7) te da je rizikvrlo visok tijekom srpnja (UVI >10). U srpnju tijekom dana UV indeks prelazi vrijednost 8 između 11 h i15 h prema lokalnom vremenu. U ovom radu za procjenu UV indeksa razvijena je jednostavna približna formula. Formula omogućava procjenu UV indeksa na temelju podataka o datumu, satu, nadmorskoj visini i naoblaci. Prilikom usporedbe rezultata dobivenih formulom i točnih rezultata dobivenih TUV modelom za ljetne mjesece lipanj, srpanj i kolovoz te razdoblje od 10 h do 16 h među rezultatima dobivena je razlikamanja od 10 %

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

Avaliação da capacidade de remoção dos íons cádmio (ii) e zinco (ii) pela macrófita egeria densa

Este trabalho avaliou a utilização da macrófita Egeria densa na remoção dosíons cádmio e zinco, em sistemas mono e bicomponente. Os experimentosforam realizados sob agitação constante, pH 4,5 e temperatura de 30 °Cpara a secagem do biossorvente e nos ensaios de biossorção. Nas condiçõesavaliadas, o equilíbrio foi atingido em cerca de 20 minutos, sendo que osdados cinéticos foram melhor descritos pelo modelo de pseudo‐segundaordem. O modelo de Langmuir modificado ajustou‐se satisfatoriamente aosdados experimentais de equilíbrio. A partir dos resultados obtidos observouseque a macrófita Egeria densa seca apresentou significativa capacidade debiossorção dos íons metálicos Cd+2 e Zn+2

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