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 <= 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

Transposable elements in Coffea (Gentianales : Rubiacea) transcripts and their role in the origin of protein diversity in flowering plants

Transposable elements are major components of plant genomes and they influence their evolution, acting as recombination hot spots, acquiring specific cell functions or becoming part of protein-coding regions. The latter is the subject of the present analysis. This study is a report on the annotation of transposable elements (TEs) in expressed sequences of Coffea arabica, Coffea canephora and Coffea racemosa, showing the occurrence of 383 ESTs and 142 unigenes with TE fragments in these three Coffea species. Based on selected unigenes, it was possible to suggest 26 putative proteins with TE-cassette insertions, demonstrating a likely contribution to protein variability. The genes for two of those proteins, the fertility restorer (FR) and the pyrophosphate-dependent phosphofructokinase (PPi-PFKs) genes, were selected for evaluating the impact of TE-cassettes on host gene evolution of other plant genomes (Arabidopsis thaliana, Oryza sativa and populus trichocarpa). This survey allowed identifying a FR gene in O. sativa harboring multiple insertions of LTR retrotransposons that originated new exons, which however does not necessarily mean a case of molecular domestication. A possible transduction event of a fragment of the PPi-PFK beta-subunit gene mediated by Helitron ATREPX1 in Arabidopsis thaliana was also highlighted

5meCpG epigenetic marks neighboring a primate-conserved core promoter short tandem repeat indicate X-chromosome inactivation.

X-chromosome inactivation (XCI) is the epigenetic transcriptional silencing of an X-chromosome during the early stages of embryonic development in female eutherian mammals. XCI assures monoallelic expression in each cell and compensation for dosage-sensitive X-linked genes between females (XX) and males (XY). DNA methylation at the carbon-5 position of the cytosine pyrimidine ring in the context of a CpG dinucleotide sequence (5meCpG) in promoter regions is a key epigenetic marker for transcriptional gene silencing. Using computational analysis, we revealed an extragenic tandem GAAA repeat 230-bp from the landmark CpG island of the human X-linked retinitis pigmentosa 2 RP2 promoter whose 5meCpG status correlates with XCI. We used this RP2 onshore tandem GAAA repeat to develop an allele-specific 5meCpG-based PCR assay that is highly concordant with the human androgen receptor (AR) exonic tandem CAG repeat-based standard HUMARA assay in discriminating active (Xa) from inactive (Xi) X-chromosomes. The RP2 onshore tandem GAAA repeat contains neutral features that are lacking in the AR disease-linked tandem CAG repeat, is highly polymorphic (heterozygosity rates approximately 0.8) and shows minimal variation in the Xa/Xi ratio. The combined informativeness of RP2/AR is approximately 0.97, and this assay excels at determining the 5meCpG status of alleles at the Xp (RP2) and Xq (AR) chromosome arms in a single reaction. These findings are relevant and directly translatable to nonhuman primate models of XCI in which the AR CAG-repeat is monomorphic. We conducted the RP2 onshore tandem GAAA repeat assay in the naturally occurring chimeric New World monkey marmoset (Callitrichidae) and found it to be informative. The RP2 onshore tandem GAAA repeat will facilitate studies on the variable phenotypic expression of dominant and recessive X-linked diseases, epigenetic changes in twins, the physiology of aging hematopoiesis, the pathogenesis of age-related hematopoietic malignancies and the clonality of cancers in human and nonhuman primates

FISH using a gag-like fragment probe reveals a common Ty3-gypsy-like retrotransposon in genome of Coffea species

The genus Coffea possesses about 100 species, and the most economically important are Coffea canephora and Coffea arabica. The latter is predominantly self-compatible with 2n = 4x = 44, while the others of the genus are diploid with 2n = 2x = 22 and mostly self-incompatible. Studies using molecular markers have been useful to detect differences between genomes in Coffea; however, molecular and cytogenetic studies have produced only limited information on the karyotypes organization. We used DOP-PCR to isolate repetitive elements from genome of Coffea arabica var. typica. The pCa06 clone, containing a fragment of 775 bp length, was characterized by sequencing and used as a probe in chromosomes of C. arabica and six other species: C. canephora, Coffea eugenioides, Coffea kapakata, Coffea liberica var. dewevrei, Coffea racemosa, and Coffea stenophylla. This insert shows similarities with a gag protein of the Ty3-gypsy-like super-family. Dot blot and FISH analyses demonstrated that pCa06 is differentially accumulated between species and chromosomes. Signals appeared scattered and clustered on the chromosomes and were also associated with heterochromatic regions. While the literature shows that there is a high karyotype similarity between Coffea species, our results point out differences in the accumulation and dispersion of this Ty3-gypsy-like retrotransposon during karyotype differentiation of Coffea.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Chromosomal and physical map positions and sequence features of the locus encompassing the <i>RP2</i> onshore tandem GAAA repeat.

<p>The composite image above the DNA sequence is based on screenshots generated using the UCSC Genome Browser (<a href="http://genome.ucsc.edu" target="_blank">http://genome.ucsc.edu</a>) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103714#pone.0103714-Kent1" target="_blank">[49]</a>, with the <i>RP2</i> onshore tandem GAAA repeat-containing region viewing coordinates chrX:46,695,746-46,696,645 (GRCh37.p5/hg19 primary reference assembly of human X-chromosome; NC_000023.10), centered on the landmark CpG island of the <i>RP2</i> promoter. The GAAA repeat element maps within the Xp11.3. The presented features (from top to bottom) are annotated tracks for OMIM genes, UCSC Genes (RefSeq, GenBank, CCDS, Rfam, tRNAs & Comparative Genomics), reference mRNA, CpG and the tandem (GAAA)n repeat. The line drawing above the DNA sequence represents the physical map of the target locus, with the <i>RP2</i> 5′coding region highlighted in light green. The locations of the forward and reverse primer sequences used for genotyping the <i>RP2</i> onshore tandem GAAA repeat are highlighted in red and pink, respectively. The tandem GAAA repeat sequence is highlighted in black with white symbols. The 5^meC-sensitive restriction endonuclease recognition sites analyzed in the XCI experiments are highlighted in blue and brown in white symbols.</p

Methylation statuses at CpG sites near the <i>RP2</i> onshore tandem GAAA repeat.

<p>Random (<b>A</b>) and non-random (<b>B</b>) X-inactivation patterns generated for different CpG-containing 5^meCpG-sensitive restriction endonuclease sites obtained using the 5^meCpG-based PCR <i>RP2</i>/<i>AR</i> biplex assay across the restriction sites. Electropherograms of alleles observed in either undigested genomic DNA or DNA digested with <i>Hpa</i>II, <i>Hha</i>I or <i>Bst</i>UI from females genotyped via quantitative fluorescent PCR are shown. The boxed numbers correspond to the areas under the allele peaks and the intensity of each peak is in relative fluorescence units (RFU).</p

<i>AR</i> CAG and the <i>RP2</i> GAAA polymorphisms refer to the same X-chromosomes.

<p>Segregation analysis of either <i>AR</i> or <i>RP2</i> alleles distinguishes the maternal origin of the preferentially skewed Xi present in the daughter. Xi is identified based on the 230-bp <i>AR</i> allele and the 368-bp <i>RP2</i> allele. The allele names are the lengths in base pairs of each fluorescence peak and the intensity of each peak is in relative fluorescence units (RFU). Note that the magnitude of stuttering at the <i>RP2</i> onshore tandem GAAA repeat is minimal, in contrast with that at the <i>AR</i> CAG repeat.</p

Molecular phylogenetic analysis using the maximum likelihood method.

<p>The evolutionary history was inferred using the maximum likelihood method based on the Tamura-Nei model <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103714#pone.0103714-Tamura2" target="_blank">[50]</a>. The bootstrap consensus tree inferred from 1,000 replicates <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103714#pone.0103714-Felsenstein1" target="_blank">[51]</a> is taken to represent the evolutionary history of the analyzed taxa <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103714#pone.0103714-Felsenstein1" target="_blank">[51]</a>. The percentages of replicate trees in which the associated taxa clustered together in the bootstrap test (1,000 replicates) are shown next to the branches <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103714#pone.0103714-Felsenstein1" target="_blank">[51]</a>. Initial tree(s) for the heuristic search were obtained automatically by applying the maximum parsimony method. The analysis involved 10 nucleotide sequences. The codon positions included were 1^st + 2^nd + 3^rd+ Noncoding. In total, there were 410 positions in the final dataset. Evolutionary analyses were conducted in MEGA5 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103714#pone.0103714-Tamura1" target="_blank">[25]</a>. The numbers in parentheses correspond to the lengths of the uninterrupted tandem arrays in GAAA repeat units.</p

The <i>RP2</i> onshore tandem GAAA repeat is polymorphic in marmosets.

<p>Electropherograms of alleles observed in marmosets genotyped via quantitative fluorescent PCR. (<b>A</b>) Representative allele profiles from males, which exhibited only the major allele, and female animals with three distinct genotypes (homozygotes for either the minor or major allele or heterozygotes) are shown. (<b>B</b>) Representative random XCI pattern observed at the 5^meCpG-sensitive <i>Bst</i>UI recognition site within the <i>RP2</i> GAAA-containing amplimer, with an Xa/Xi ratio of approximately 65%. The allele names are the lengths in base pairs of each fluorescence peak and the intensity of each peak is in relative fluorescence units (RFU).</p

Reverse transcription-PCR across the GAAA repeat-containing region.

<p><i>RP2</i> onshore tandem GAAA repeat-specific steady-state RNA is not detected in mononucleated blood cells from two healthy female donors (21 years old) or a male donor (33 years old) or from the THP-1 male cell line. RNA samples were either reverse (+) or mock (−) transcribed (RT) prior to PCR amplification across the <i>RP2</i> onshore tandem GAAA repeat-specific region (<b>A</b>) or the <i>GAPDH</i>-specific region (<b>B</b>). Corresponding samples of genomic DNA were used as positive controls for the PCR assays. The amplification products were separated via electrophoresis in an 8% acrylamide: bis-acrylamide gel and silver-stained for detection. Lane L50 shows a standard 50-bp ladder (Invitrogen); lane H₂O is the negative PCR amplification control. The range of the <i>RP2</i> onshore tandem GAAA repeat-specific DNA amplimer is 350 to 391-bp. The <i>GAPDH</i>-specific DNA amplimers are as follows: 130-bp for <i>GAPDHP63</i> (6∶80663360-80663489) and <i>GAPDHP1</i> (X:39647022-39647151) and 220-bp for <i>GAPDH</i> (12∶6646089-6646308). The processed (mature) <i>GAPDH</i>-specific cDNA-derived product is 130- bp.</p