Sox2 is important for two crucial processes in lung development: Branching morphogenesis and epithelial cell differentiation

AbstractThe primary lung bud originates from the foregut and develops into the bronchial tree by repetitive branching and outgrowing of the airway. The Sry related HMG box protein Sox2 is expressed in a cyclic manner during initiation and branching morphogenesis of the lung. It is highly expressed in non-branching regions and absent from branching regions, suggesting that downregulation of Sox2 is mandatory for airway epithelium to respond to branch inducing signals. Therefore, we developed transgenic mice that express a doxycycline inducible Sox2 in the airway epithelium. Continuous expression of Sox2 hampers the branching process resulting in a severe reduction of the number of airways. In addition, the bronchioli transiently go over into enlarged, alveolar-like airspaces, a pathology described as bronchiolization of alveoli. Furthermore, a substantial increase was observed of cGRP positive neuroendocrine cells and ΔNp63 isoform expressing (pre-) basal cells, which are both committed precursor-like cells. Thus, Sox2 prevents airways from branching and prematurely drives cells into committed progenitors, apparently rendering these committed progenitors unresponsive to branch inducing signals. However, Sox2 overexpression does not lead to a complete abrogation of the epithelial differentiation program

Short and long-read ultra-deep sequencing profiles emerging heterogeneity across five platform Escherichia coli strains

Reprogramming organisms for large-scale bioproduction counters their evolutionary objectives of fast growth and often leads to mutational collapse of the engineered production pathways during cultivation. Yet, the mutational susceptibility of academic and industrial Escherichia coli bioproduction host strains are poorly understood. In this study, we apply 2nd and 3rd generation deep sequencing to profile simultaneous modes of genetic heterogeneity that decimate engineered biosynthetic production in five popular E. coli hosts BL21(DE3), TOP10, MG1655, W, and W3110 producing 2,3-butanediol and mevalonic acid. Combining short-read and longread sequencing, we detect strain and sequence-specific mutational modes including single nucleotide polymorphism, inversion, and mobile element transposition, as well as complex structural variations that disrupt the integrity of the engineered biosynthetic pathway. Our analysis suggests that organism engineers should avoid chassis strains hosting active insertion sequence (IS) subfamilies such as IS1 and IS10 present in popular E. coli TOP10. We also recommend monitoring for increased mutagenicity in the pathway transcription initiation regions and recombinogenic repeats. Together, short and long sequencing reads identified latent low-frequency mutation events such as a short detrimental inversion within a pathway gene, driven by 8-bp short inverted repeats. This demonstrates the power of combining ultra-deep DNA sequencing technologies to profile genetic heterogeneities of engineered constructs and explore the markedly different mutational landscapes of common E. coli host strains. The observed multitude of evolving variants underlines the usefulness of early mutational profiling for new synthetic pathways designed to sustain in organisms over long cultivation scales

TIDLIG INTERVENTION I SPÆDBARNSFAMILIER - Forebyggende rådgivning og behandling gennem tre årtier

Med udgangspunkt i den kliniske erfaring og forskning, der er opnået ved Center for Spædbørn (CFS) igennem de sidste årtier, vil nærværende artikel reflektere over centrale aspekter ved tidlig intervention i nyblevne familier. På baggrund af en større undersøgelse foretaget i CFS, fremhæves det, at den tidlige intervention i nyblevne familier ikke kun skal rettes imod traditionelt definerede risikofamilier, men at støtten skal tilbydes bredt. I behandlingsarbejdet med spædbørnsfamilier argumenteres der for at anskue familiens vanskeligheder ud fra et normalpsykologisk udgangspunkt, hvor en gennemgribende psykologisk forandringsproces synes at være en almengyldig bestanddel af forældreudviklingen hos begge køn. Følgelig anses det for hensigtsmæssigt, at behandlingen rettes mod morfar-spædbarn-triaden, da adskillige undersøgelser omkring faderskabet understreger den opfattelse, at begge køn ved et spædbarns fødsel er i en sensitiv og sårbar periode, hvor selv tilsyneladende mindre vanskeligheder kan udløse store problemer, som ofte også udspiller sig i eller belaster parforholdet. Endelig betones vigtigheden af at inddrage spædbarnet i behandlingen, da spædbarnet giver den trænede observatør informationer ikke blot om dets egen tilstand, men også er en væsentlig kilde til information om forældrenes ressourcer og samspil med barnet. Ud fra CFS’s erfaring har spædbarnets tilstedeværelse i behandlingsarbejdet en faciliterende og motiverende indflydelse på den samlede families udviklingsproces og udbytte af interventionen

Differentiated type II pneumocytes can be reprogrammed by ectopic Sox2 expression

The adult lung contains several distinct stem cells, although their properties and full potential are still being sorted out. We previously showed that ectopic Sox2 expression in the developing lung manipulated the fate of differentiating cells. Here, we addressed the question whether fully differentiated cells could be redirected towards another cell type. Therefore, we used transgenic mice to express an inducible Sox2 construct in type II pneumocytes, which are situated in the distal, respiratory areas of the lung. Within three days after the induction of the transgene, the type II cells start to proliferate and form clusters of cuboidal cells. Prolonged Sox2 expression resulted in the reversal of the type II cell towards a more embryonic, precursor-like cell, being positive for the stem cell markers Sca1 and Ssea1. Moreover, the cells started to co-express Spc and Cc10, characteristics of bronchioalveolar stem cells. We demonstrated that Sox2 directly regulates the expression of Sca1. Subsequently, these cells expressed Trp63, a marker for basal cells of the trachea. So, we show that the expression of one transcription factor in fully differentiated, distal lung cells changes their fate towards proximal cells through intermediate cell types. This may have implications for regenerative medicine, and repair of diseased and damaged lungs

Hypoxia Inducible Factor 3α Plays a Critical Role in Alveolarization and Distal Epithelial Cell Differentiation during Mouse Lung Development

Lung development occurs under relative hypoxia and the most important oxygen-sensitive response pathway is driven by Hypoxia Inducible Factors (HIF). HIFs are heterodimeric transcription factors of an oxygen-sensitive subunit, HIFα, and a constitutively expressed subunit, HIF1β. HIF1α and HIF2α, encoded by two separate genes, contribute to the activation of hypoxia inducible genes. A third HIFα gene, HIF3α, is subject to alternative promoter usage and splicing, leading to three major isoforms, HIF3α, NEPAS and IPAS. HIF3α gene products add to the complexity of the hypoxia response as they function as dominant negative inhibitors (IPAS) or weak transcriptional activators (HIF3α/NEPAS). Previously, we and others have shown the importance of the Hif1α and Hif2α factors in lung development, and here we investigated the role of Hif3α during pulmonary development. Therefore, HIF3α was conditionally expressed in airway epithelial cells during gestation and although HIF3α transgenic mice were born alive and appeared normal, their lungs showed clear abnormalities, including a post-pseudoglandular branching defect and a decreased number of alveoli. The HIF3α expressing lungs displayed reduced numbers of Clara cells, alveolar epithelial type I and type II cells. As a result of HIF3α expression, the level of Hif2α was reduced, but that of Hif1α was not affected. Two regulatory genes, Rarβ, involved in alveologenesis, and Foxp2, a transcriptional repressor of the Clara cell specific Ccsp gene, were significantly upregulated in the HIF3α expressing lungs. In addition, aberrant basal cells were observed distally as determined by the expression of Sox2 and p63. We show that Hif3α binds a conserved HRE site in the Sox2 promoter and weakly transactivated a reporter construct containing the Sox2 promoter region. Moreover, Hif3α affected the expression of genes not typically involved in the hypoxia response, providing evidence for a novel function of Hif3α beyond the hypoxia response

La communication sous forme d’un jeu de plateau pour partager des données et des ressentis d’experts à propos d’un nouveau traitement contre le cancer

La conception de cartes à des fins de communication ou, de manière plus générale, la visualisation de données est un champ de recherches relativement ancien qui a subi de profonds changements au cours de ces dernières années. Si de nombreuses recherches y sont consacrées, très peu d’entre elles s’intéressent à une autre tendance forte du moment : l’emploi ou le détournement du jeu à des fins de communication. Ce papier traite de cette question en abordant le cas d’un support informationnel rappelant un jeu de stratégie sur plateau développé dans un but d’information à propos d’un nouveau procédé de lutte contre le cancer : la thérapie photodynamique.La conception de cartes à des fins de communication ou, de manière plus générale, la visualisation de données est un champ de recherches relativement ancien qui a subi de profonds changements au cours de ces dernières années. Si de nombreuses recherches y sont consacrées, très peu d’entre elles s’intéressent à une autre tendance forte du moment : l’emploi ou le détournement du jeu à des fins de communication. Ce papier traite de cette question en abordant le cas d’un support informationnel rappelant un jeu de stratégie sur plateau développé dans un but d’information à propos d’un nouveau procédé de lutte contre le cancer : la thérapie photodynamique

Unique tracheal fluid microRNA signature predicts response to FETO in patients with congenital diaphragmatic hernia

"Epub ahead of print 2015 Jan 5"OBJECTIVE AND BACKGROUND: Our objective was to determine the fetal in vivo microRNA signature in hypoplastic lungs of human fetuses with severe isolated congenital diaphragmatic hernia (CDH) and changes in tracheal and amniotic fluid of fetuses undergoing fetoscopic endoluminal tracheal occlusion (FETO) to reverse severe lung hypoplasia due to CDH. METHODS:: We profiled microRNA expression in prenatal human lungs by microarray analysis. We then validated this signature with real-time quantitative polymerase chain reaction in tracheal and amniotic fluid of CDH patients undergoing FETO. We further explored the role of miR-200b using semiquantitative in situ hybridization and immunohistochemistry for TGF-ß2 in postnatal lung sections. We investigated miR-200b effects on TGF-ß signaling using a SMAD-luciferase reporter assay and Western blotting for phospho-SMAD2/3 and ZEB-2 in cultures of human bronchial epithelial cells. RESULTS:: CDH lungs display an increased expression of 2 microRNAs: miR-200b and miR-10a as compared to control lungs. Fetuses undergoing FETO display increased miR-200 expression in their tracheal fluid at the time of balloon removal. Future survivors of FETO display significantly higher miR-200 expression than those with a limited response. miR-200b was expressed in bronchial epithelial cells and vascular endothelial cells. TGF-ß2 expression was lower in CDH lungs. miR-200b inhibited TGF-ß-induced SMAD signaling in cultures of human bronchial epithelial cells. CONCLUSIONS:: Human fetal hypoplastic CDH lungs have a specific miR-200/miR-10a signature. Survival after FETO is associated with increased miR-200 family expression. miR-200b overexpression in CDH lungs results in decreased TGF-ß/SMAD signaling

Selection of potential targets for stratifying congenital pulmonary airway malformation patients with molecular imaging:is MUC1 the one?

Currently there is a global lack of consensus about the best treatment for asymptomatic congenital pulmonary airway malformation (CPAM) patients. The somatic KRAS mutations commonly found in adult lung cancer combined with mucinous proliferations are sometimes found in CPAM. For this risk of developing malignancy, 70% of paediatric surgeons perform a resection for asymptomatic CPAM. In order to stratify these patients into high-and low-risk groups for developing malignancy, a minimally invasive diagnostic method is needed, for example targeted molecular imaging. A prerequisite for this technique is a cell membrane bound target. The aim of this study was to review the literature to identify potential targets for molecular imaging in CPAM patients and perform a first step to validate these findings. A systematic search was conducted to identify possible targets in CPAM and adenocarcinoma in situ (AIS) patients. The most interesting targets were evaluated with immunofluorescent staining in adjacent lung tissue, KRAS+ CPAM tissue and KRAS– CPAM tissue. In 185 included studies, 143 possible targets were described, of which 20 targets were upregulated and membrane-bound. Six of them were also upregulated in lung AIS tissue (CEACAM5, E-cadherin, EGFR, ERBB2, ITGA2 and MUC1) and as such of possible interest. Validating studies showed that MUC1 is a potential interesting target. This study provides an extensive overview of all known potential targets in CPAM that might identify those patients at risk for malignancy and conducted the first step towards validation, identifying MUC1 as the most promising target.</p