Background: The purpose of this study is to provide evidence-based and expert consensus recommendations for lung ultrasound with focus on emergency and critical care settings. Methods: A multidisciplinary panel of 28 experts from eight countries was involved. Literature was reviewed from January 1966 to June 2011. Consensus members searched multiple databases including Pubmed, Medline, OVID, Embase, and others. The process used to develop these evidence-based recommendations involved two phases: determining the level of quality of evidence and developing the recommendation. The quality of evidence is assessed by the grading of recommendation, assessment, development, and evaluation (GRADE) method. However, the GRADE system does not enforce a specific method on how the panel should reach decisions during the consensus process. Our methodology committee decided to utilize the RAND appropriateness method for panel judgment and decisions/consensus. Results: Seventythree proposed statements were examined and discussed in three conferences held in Bologna, Pisa, and Rome. Each conference included two rounds of face-to-face modified Delphi technique. Anonymous panel voting followed each round. The panel did not reach an agreement and therefore did not adopt any recommendations for six statements. Weak/ conditional recommendations were made for 2 statements, and strong recommendations were made for the remaining 65 statements. The statements were then recategorized and grouped to their current format. Internal and external peer-review processes took place before submission of the recommendations. Updates will occur at least every 4 years or whenever significant major changes in evidence appear. Conclusions: This document reflects the overall results of the first consensus conference on ''point-of-care'' lung ultrasound. Statements were discussed and elaborated by experts who published the vast majority of papers on clinical use of lung ultrasound in the last 20 years. Recommendations were produced to guide implementation, development, and standardization of lung ultrasound in all relevant settings.

Nanog is a divergent homeodomain protein found in mammalian pluripotent cells and developing germ cells. Deletion of Nanog causes early embryonic lethality, whereas constitutive expression enables autonomous self-renewal of embryonic stem cells. Nanog is accordingly considered a core element of the pluripotent transcriptional network. However, here we report that Nanog fluctuates in mouse embryonic stem cells. Transient downregulation of Nanog appears to predispose cells towards differentiation but does not mark commitment. By genetic deletion we show that, although they are prone to differentiate, embryonic stem cells can self-renew indefinitely in the permanent absence of Nanog. Expanded Nanog null cells colonize embryonic germ layers and exhibit multilineage differentiation both in fetal and adult chimaeras. Although they are also recruited to the germ line, primordial germ cells lacking Nanog fail to mature on reaching the genital ridge. This defect is rescued by repair of the mutant allele. Thus Nanog is dispensible for expression of somatic pluripotency but is specifically required for formation of germ cells. Nanog therefore acts primarily in construction of inner cell mass and germ cell states rather than in the housekeeping machinery of pluripotency. We surmise that Nanog stabilizes embryonic stem cells in culture by resisting or reversing alternative gene expression states.

SummaryPluripotency is generated naturally during mammalian development through formation of the epiblast, founder tissue of the embryo proper. Pluripotency can be recreated by somatic cell reprogramming. Here we present evidence that the homeodomain protein Nanog mediates acquisition of both embryonic and induced pluripotency. Production of pluripotent hybrids by cell fusion is promoted by and dependent on Nanog. In transcription factor-induced molecular reprogramming, Nanog is initially dispensable but becomes essential for dedifferentiated intermediates to transit to ground state pluripotency. In the embryo, Nanog specifically demarcates the nascent epiblast, coincident with the domain of X chromosome reprogramming. Without Nanog, pluripotency does not develop, and the inner cell mass is trapped in a pre-pluripotent, indeterminate state that is ultimately nonviable. These findings suggest that Nanog choreographs synthesis of the naive epiblast ground state in the embryo and that this function is recapitulated in the culmination of somatic cell reprogramming.

In many higher organisms, 5%-15% of histone H2A is ubiquitylated at lysine 119 (uH2A). The function of this modification and the factors involved in its establishment, however, are unknown. Here we demonstrate that uH2A occurs on the inactive X chromosome in female mammals and that this correlates with recruitment of Polycomb group (PcG) proteins belonging to Polycomb repressor complex 1 (PRC1). Based on our observations, we tested the role of the PRC1 protein Ring1B and its closely related homolog Ring1A in H2A ubiquitylation. Analysis of Ring1B null embryonic stem (ES) cells revealed extensive depletion of global uH2A levels. On the inactive X chromosome, uH2A was maintained in Ring1A or Ring1B null cells, but not in double knockout cells, demonstrating an overlapping function for these proteins in development. These observations link H2A ubiquitylation, X inactivation, and PRC1 PcG function, suggesting an unanticipated and novel mechanism for chromatin-mediated heritable gene silencing.

Embryonic stem (ES) cells have been available from inbred mice since 1981 but have not been validated for other rodents. Failure to establish ES cells from a range of mammals challenges the identity of cultivated stem cells and our understanding of the pluripotent state. Here we investigated derivation of ES cells from the rat. We applied molecularly defined conditions designed to shield the ground state of authentic pluripotency from inductive differentiation stimuli. Undifferentiated cell lines developed that exhibited diagnostic features of ES cells including colonization of multiple tissues in viable chimeras. Definitive ES cell status was established by transmission of the cell line genome to offspring. Derivation of germline-competent ES cells from the rat paves the way to targeted genetic manipulation in this valuable biomedical model species. Rat ES cells will also provide a refined test-bed for functional evaluation of pluripotent stem cell-derived tissue repair and regeneration.

Summary By analyzing gene expression data in gliobastoma in combination with matched microRNA profiles, we have uncovered a post-transcriptional regulation layer of surprising magnitude, comprising over 248,000 microRNA (miR)-mediated interactions. These include ~7,000 genes whose transcripts act as miR ‘sponges’ and 148 genes that act through alternative, non-sponge interactions. Biochemical analyses in cell lines confirmed that this network regulates established drivers of tumor initiation and subtype, including PTEN, PDGFRA, RB1, VEGFA, STAT3, and RUNX1, suggesting that these interactions mediate crosstalk between canonical oncogenic pathways. RNA silencing of 13 microRNA-mediated PTEN regulators, whose locus deletions are predictive of PTEN expression variability, was sufficient to downregulate PTEN in a 3′ UTR-dependent manner and to increase tumor-cell growth rates. Thus, this miR-mediated network provides a mechanistic, experimentally validated rationale for the loss of PTEN expression in a large number of glioma samples with an intact PTEN locus.

Previous studies have implicated the Eed-Enx1 Polycomb group complex in the maintenance of imprinted X inactivation in the trophectoderm lineage in mouse. Here we show that recruitment of Eed-Enx1 to the inactive X chromosome (Xi) also occurs in random X inactivation in the embryo proper. Localization of Eed-Enx1 complexes to Xi occurs very early, at the onset of Xist expression, but then disappears as differentiation and development progress. This transient localization correlates with the presence of high levels of the complex in totipotent cells and during early differentiation stages. Functional analysis demonstrates that Eed-Enx1 is required to establish methylation of histone H3 at lysine 9 and/or lysine 27 on Xi and that this, in turn, is required to stabilize the Xi chromatin structure.

Embryonic stem (ES) cells can be derived and propagated from multiple strains of mouse and rat through application of smallmolecule inhibitors of the fibroblast growth factor (FGF)/Erk pathway and of glycogen synthase kinase 3. These conditions shield pluripotent cells from differentiation-inducing stimuli. We investigate the effect of these inhibitors on the development of pluripotent epiblast in intact pre-implantation embryos. We find that blockade of Erk signalling from the 8-cell stage does not impede blastocyst formation but suppresses development of the hypoblast. The size of the inner cell mass (ICM) compartment is not reduced, however. Throughout the ICM, the epiblast-specific marker Nanog is expressed, and in XX embryos epigenetic silencing of the paternal X chromosome is erased. Epiblast identity and pluripotency were confirmed by contribution to chimaeras with germline transmission. These observations indicate that segregation of hypoblast from the bipotent ICM is dependent on FGF/Erk signalling and that in the absence of this signal, the entire ICM can acquire pluripotency. Furthermore, the epiblast does not require paracrine support from the hypoblast. Thus, naïve epiblast and ES cells are in a similar ground state, with an autonomous capacity for survival and replication, and high vulnerability to Erk signalling. We probed directly the relationship between naïve epiblast and ES cells. Dissociated ICM cells from freshly harvested late blastocysts gave rise to up to 12 ES cell clones per embryo when plated in the presence of inhibitors. We propose that ES cells are not a tissue culture creation, but are essentially identical to preimplantation epiblast cells.