Aberrant DNA methylation and expression of SPDEF and FOXA2 in airway epithelium of patients with COPD.

BACKGROUND: Goblet cell metaplasia, a common feature of chronic obstructive pulmonary disease (COPD), is associated with mucus hypersecretion which contributes to the morbidity and mortality among patients. Transcription factors SAM-pointed domain-containing Ets-like factor (SPDEF) and forkhead box protein A2 (FOXA2) regulate goblet cell differentiation. This study aimed to (1) investigate DNA methylation and expression of SPDEF and FOXA2 during goblet cell differentiation and (2) compare this in airway epithelial cells from patients with COPD and controls during mucociliary differentiation. METHODS: To assess DNA methylation and expression of SPDEF and FOXA2 during goblet cell differentiation, primary airway epithelial cells, isolated from trachea (non-COPD controls) and bronchial tissue (patients with COPD), were differentiated by culture at the air-liquid interface (ALI) in the presence of cytokine interleukin (IL)-13 to promote goblet cell differentiation. RESULTS: We found that SPDEF expression was induced during goblet cell differentiation, while FOXA2 expression was decreased. Importantly, CpG number 8 in the SPDEF promoter was hypermethylated upon differentiation, whereas DNA methylation of FOXA2 promoter was not changed. In the absence of IL-13, COPD-derived ALI-cultured cells displayed higher SPDEF expression than control-derived ALI cultures, whereas no difference was found for FOXA2 expression. This was accompanied with hypomethylation of CpG number 6 in the SPDEF promoter and also hypomethylation of CpG numbers 10 and 11 in the FOXA2 promoter. CONCLUSIONS: These findings suggest that aberrant DNA methylation of SPDEF and FOXA2 is one of the factors underlying mucus hypersecretion in COPD, opening new avenues for epigenetic-based inhibition of mucus hypersecretion.

Prenatal exposure to tobacco smoke sex dependently influences methylation and mRNA levels of the Igf axis in lungs of mouse offspring.

Prenatal smoke exposure is a risk factor for abnormal lung development and increased sex-dependent susceptibility for asthma and chronic obstructive pulmonary disease (COPD). Birth cohort studies show genome-wide DNA methylation changes in children from smoking mothers, but evidence for sex-dependent smoke-induced effects is limited. The insulin-like growth factor (IGF) system plays an important role in lung development. We hypothesized that prenatal exposure to smoke induces lasting changes in promoter methylation patterns of Igf1 and Igf1r, thus influencing transcriptional activity and contributing to abnormal lung development. We measured and compared mRNA levels along with promoter methylation of Igf1 and Igf1r and their protein concentrations in lung tissue of 30-day-old mice that had been prenatally exposed to cigarette smoke (PSE) or filtered air (control). Body weight at 30 days after birth was measured as global indicator of normal development. Female PSE mice showed lower mRNA levels of Igf1 and its receptor (Igf1: P = 0.05; Igf1r: P = 0.03). Furthermore, CpG-site-specific methylation changes were detected in Igf1r in a sex-dependent manner and the body weight of female offspring was reduced after prenatal exposure to smoke, while protein concentrations were unaffected. Prenatal exposure to smoke induces a CpG-site-specific loss of Igf1r promoter methylation, which can be associated with body weight. These findings highlight the sex-dependent and potentially detrimental effects of in utero smoke exposure on DNA methylation and Igf1 and Igf1r mRNA levels. The observations support a role for Igf1 and Igf1r in abnormal development.

Diversity and temporal patterns of planktonic protist assemblages at a Mediterranean Long Term Ecological Research site.

We tracked temporal changes in protist diversity at the Long Term Ecological Research (LTER) station MareChiara in the Gulf of Naples (Mediterranean Sea) on eight dates in 2011 using a metabarcoding approach. Illumina analysis of the V4 and V9 fragments of the 18S rDNA produced 869 522 and 1 410 071 sequences resulting in 6517 and 6519 OTUs, respectively. Marked compositional variations were recorded across the year, with less than 2% of OTUs shared among all samples and similar patterns for the two marker tags. Alveolata, Stramenopiles and Rhizaria were the most represented groups. A comparison with light microscopy data indicated an over-representation of Dinophyta in the sequence dataset, whereas Bacillariophyta showed comparable taxonomic patterns between sequence and light microscopy data. Shannon diversity values were stable from February to September, increasing thereafter with a peak in December. Community variance was mainly explained by seasonality (as temperature), trophic status (as chlorophyll a), and influence of coastal waters (as salinity). Overall, the background knowledge of the system provided a sound context for the result interpretation, showing that LTER sites provide an ideal setting for high-throughput sequencing (HTS) metabarcoding characterisation of protist assemblages and their relationships with environmental variations.

[Serious intoxication with hydroxychloroquine, with haemodynamic instability: a case report supporting treatment with diazepam]. / Ernstige intoxicatie met hydroxychloroquine, met hemodynamische instabiliteit: casuistische ondersteuning voor behandeling met diazepam.

A 37-year-old woman was admitted to the emergency room because of an autointoxication with hydroxychloroquine, leading to haemodynamic instability. Treatment consisted of the rapid administration of intravenous diazepam, after which the hypotension recovered rapidly even though no vasoactive medication was given. Treatment with diazepam has been advised in the Netherlands for many years in case of severe hydroxychloroquine intoxication, despite the fact that convincing evidence for its use is lacking. On the basis of the experience with the relevant cases, the administration of diazepam, 2 mg/kg initially followed by 2 mg/kg/24 hours as a continuous infusion, should certainly be considered for supportive treatment in the ICU in case of severe haemodynamic instability.

[Again new resuscitation guidelines (2006): justification, costs and potential confusion]. / Weer nieuwe richtlijnen voor reanimatie (2006): onderbouwing, kosten en mogelijke verwarring.

The last revision of the Dutch resuscitation guidelines, a translation of the European Resuscitation Council Guidelines 2005, is based on the recommendations of the International Liaison Committee on Resuscitation (ILCOR). The previous Dutch guidelines were issued in 2002. Most changes are based on laboratory studies and retrospective analyses. The most important changes are: recognizing circulatory arrest on unresponsiveness and abnormal breathing; a new ratio of chest compressions to ventilations i.e. 30:2 instead of 15:2; and following the procedure of checking the airway (A), taking over the circulation (C) and breathing (B). Furthermore in the event of ventricular fibrillation or ventricular tachycardia with no pulsations then one defibrillator shock only is to be given; this is in contrast with the previous application of cycles of 3 shocks. The work and costs of implementation involved in the revision of resuscitation guidelines are tremendous, especially in view of the huge number of laypersons who need to be retrained. Also, frequent changes of guidelines may cause confusion and have a negative effect on the quality of resuscitation. Therefore, it is not evident that the benefits of this revision justify its costs. It would be good to prospectively evaluate the effectiveness and costs of this revision. In the future, these data might help to decide when altered international recommendations should be translated into new Dutch resuscitation guidelines. Alternative strategies should be considered, for example only changing the guidelines for advanced life support.

Life-threatening Pneumocystis jiroveci pneumonia following treatment of severe Cushing's syndrome.

We describe two patients with a severe Cushing's syndrome due to ectopic production of ACTH. Both patients developed a life-threatening Pneumocystis jiroveci pneumonia (PCP) shortly after treatment of the hypercortisolism was started by means of inhibition of production of glucocorticoids and glucocorticoid receptor blockade. We presume that the restored immune response elicited the clinical symptoms of the opportunistic, previously subclinical Pneumocystis jiroveci infection. The immunocompromised state and the delicate glucocorticoid balance in patients with a severe Cushing's syndrome necessitate a specific diagnostic and therapeutic approach.

Ribosomal RNA analysis indicates a benthic pennate diatom ancestry for the endosymbionts of the dinoflagellates Peridinium foliaceum and Peridinium balticum (Pyrrhophyta).

The establishment of chloroplasts as cellular organelles in the dinoflagellate, heterokont (stramenopile), haptophyte, and cryptophyte algae is widely accepted to have been the result of secondary endosymbiotic events, that is, the uptake of a photosynthetic eukaryote by a phagotrophic eukaryote. However, the circumstances that promote such associations between two phylogenetically distinct organisms and result in the integration of their genomes to form a single functional photosynthetic cell is unclear. The dinoflagellates Peridinium foliaceum and Peridinium balticum are unusual in that each contains a membrane-bound eukaryotic heterokont endosymbiont. These symbioses have been interpreted, through data derived from ultrastructural and biochemical investigations, to represent an intermediate stage of secondary endosymbiotic chloroplast acquisition. In this study we have examined the phylogenetic origin of the P. foliaceum and P. balticum heterokont endosymbionts through analysis of their nuclear small subunit ribosomal RNA genes. Our analyses clearly demonstrate both endosymbionts are pennate diatoms belonging to the family Bacillariaceae. Since members of the Bacillariaceae are usually benthic, living on shallow marine sediments, the manner in which establishment of a symbiosis between a planktonic flagellated dinoflagellate and a bottom-dwelling diatom is discussed. In particular, specific environmentally-associated life strategy stages of the host and symbiont, coupled with diatom food preferences by the dinoflagellate, may have been vital to the formation of this association.

Evolution of the diatoms (Bacillariophyta). IV. A reconstruction of their age from small subunit rRNA coding regions and the fossil record.

Small subunit ribosomal RNA (ssu rRNA) coding regions from 30 diatoms, 3 oomycetes, and 6 pelagophytes were used to construct linearized trees, maximum-likelihood trees, and neighbor-joining trees inferred from both unweighted and weighted distances. Stochastic accumulation of sequence substitutions among the diatoms was assessed with relative rate tests. Pennate diatoms evolved relatively slowly but within the limits set by a stochastic model; centric diatoms exceeded those limits. A rate distribution test was devised to identify those taxa showing an aberrant distribution of base substitutions within the ssu rRNA coding region. First appearance dates of diatom taxa from the fossil record were regressed against their corresponding branch lengths to infer the average and earliest possible age for the origin of the diatoms, the pennate diatoms, and the centric diatom order Thalassiosirales. Our most lenient age estimate (based on the median-evolving diatom taxon in the maximum-likelihood tree or on the average branch length in a linearized tree) suggests that their average age is approximately 164-166 Ma, which is close to their earliest fossil record. Both calculations suggest that it is unlikely that diatoms existed prior to 238-266 Ma. Rate variation among the diatoms' ssu rRNA coding regions and uncertainties associated with the origin of extant taxa in the fossil record contribute significantly to the variation in age estimates obtained. Different evolutionary models and the exclusion of fast or slow evolving taxa did not significantly affect age estimates; however, the inclusion of aberrantly fast evolving taxa did. Our molecular clock calibrations indicate that the rRNA coding regions in the diatoms are evolving at approximately 1% per 18 to 26 Ma, which is the fastest substitution rate reported in any pro- or eukaryotic group of organisms to date.

Evolution of the diatoms (Bacillariophyta). II. Nuclear-encoded small-subunit rRNA sequence comparisons confirm a paraphyletic origin for the centric diatoms.

A phylogeny of the diatoms was inferred from comparisons of nuclear-encoded small subunit ribosomal RNA coding regions using maximum likelihood, weighted maximum parsimony, and neighbor-joining distance methods with Jukes and Cantor, Kimura, Gamma, van de Peer, and LogDet evolutionary models. Analyses of 30 taxa in 11 orders recovered two clades (Clades I and II). Neither of these clades correspond to the three classes of diatoms presently recognized or to the traditionally recognized radially symmetrical centric diatoms or bilaterally symmetrical pennate diatoms. All analyses show that the centric diatoms are a paraphyletic lineage. Tests of alternative phylogenies that address existing hypotheses regarding diatom systematics with the maximum likelihood and maximum parsimony methods support the two clades. Clade I is defined by centric diatom orders with specialized tubes, termed labiate processes, located peripherally in the cell wall. Clade II contains (1) bi(multi)polar centric diatoms with centrally located labiate processes, (2) centric diatoms with other central tubes termed strutted processes, and (3) pennate diatoms. Morphological evidence from fossil assemblages and cytological architecture support the results of the molecular analyses, whereas morphological features of extant diatoms are too derived to resolve the deeper branches in the tree.