New insights into the Usnea cornuta aggregate (Parmeliaceae, lichenized Ascomycota): Molecular analysis reveals high genetic diversity correlated with chemistry.

Biological processes such as hybridization, incomplete lineage sorting and gene flow can obscure the recognition of distinct evolutionary lineages, particularly in groups of organisms that have recently diverged. Therefore, compiling pieces of evidence from diverse data sources is critical to accurately assess species boundaries in such groups. The increasing availability of DNA sequence data allows for a much deeper understanding of diversification and speciation processes and their consequences on biodiversity. In this study, we applied an integrative approach based on DNA sequence, chemical, geographic and morphological data to attempt to define species boundaries in the lichen-forming genus Usnea (Parmeliaceae), particularly the U. cornuta aggregate, a cosmopolitan species group. We provide the first species delimitation for this group in the neotropics based on the multispecies coalescent (MSC) model. Using ITS rDNA and two protein-coding genes, Mcm7 and RPB1, we estimated the species tree under the MSC model in a Bayesian framework using STACEY. Our results indicate that at least nine strongly supported distinct lineages coexist in the U. cornuta aggregate, which are well chemically characterized. Additionally, we found evidence for the polyphyly of three morphospecies, Usnea brasiliensis, U. cornuta and U. dasaea.

Preparation and optimization of pyrazolo[1,5-a]pyrimidines as new potent PDE4 inhibitors.

A new series of pyrazolo[1,5-a]pyrimidines exemplified by compound 1, has been identified with moderate activity (IC50=165nM), following GSK256066 rescaffolding. Compound 1 optimization at positions 2, 3, 6 and 7 gave compound 10 with high in vitro activity (IC50=0.7nM). Modeling studies based on the PDB structure 3GWT with compound 5 showed the expected overlay with the carboxamide, the aryl moiety and the sulfone. Cyclisation of the primary amide to the 5 position of the pyrazolo[1,5-a]pyrimidines scaffold afforded compounds 15 and 16 with 200-fold enhancement in activity and cellular potency.

Synthesis and multiparametric evaluation of thiadiazoles and oxadiazoles as diacylglycerol acyltransferase type 1 inhibitors.

Chemical modulation of a formerly disclosed DGAT-1 inhibitor resulted in the identification of a compound with a suitable profile for preclinical development. Optimisation of solubility is discussed and a PK/PD study is presented.

Evolution of complex symbiotic relationships in a morphologically derived family of lichen-forming fungi.

We studied the evolutionary history of the Parmeliaceae (Lecanoromycetes, Ascomycota), one of the largest families of lichen-forming fungi with complex and variable morphologies, also including several lichenicolous fungi. We assembled a six-locus data set including nuclear, mitochondrial and low-copy protein-coding genes from 293 operational taxonomic units (OTUs). The lichenicolous lifestyle originated independently three times in lichenized ancestors within Parmeliaceae, and a new generic name is introduced for one of these fungi. In all cases, the independent origins occurred c. 24 million yr ago. Further, we show that the Paleocene, Eocene and Oligocene were key periods when diversification of major lineages within Parmeliaceae occurred, with subsequent radiations occurring primarily during the Oligocene and Miocene. Our phylogenetic hypothesis supports the independent origin of lichenicolous fungi associated with climatic shifts at the Oligocene-Miocene boundary. Moreover, diversification bursts at different times may be crucial factors driving the diversification of Parmeliaceae. Additionally, our study provides novel insight into evolutionary relationships in this large and diverse family of lichen-forming ascomycetes.

Testing the use of ITS rDNA and protein-coding genes in the generic and species delimitation of the lichen genus Usnea (Parmeliaceae, Ascomycota).

In lichen-forming fungi, traditional taxonomical concepts are frequently in conflict with molecular data, and identifying appropriate taxonomic characters to describe phylogenetic clades remains challenging in many groups. The selection of suitable markers for the reconstruction of solid phylogenetic hypotheses is therefore fundamental. The lichen genus Usnea is highly diverse, with more than 350 estimated species, distributed in polar, temperate and tropical regions. The phylogeny and classification of Usnea have been a matter of debate, given the lack of phenotypic characters to describe phylogenetic clades and the low degree of resolution of phylogenetic trees. In this study, we investigated the phylogenetic relationships of 52 Usnea species from across the genus, based on ITS rDNA, nuLSU, and two protein-coding genes RPB1 and MCM7. ITS comprised several highly variable regions, containing substantial genetic signal, but also susceptible to causing bias in the generation of the alignment. We compared several methods of alignment of ITS and found that a simultaneous optimization of alignment and phylogeny (using BAli-phy) improved significantly both the topology and the resolution of the phylogenetic tree. However the resolution was even better when using protein-coding genes, especially RPB1 although it is less variable. The phylogeny based on the concatenated dataset revealed that the genus Usnea is subdivided into four highly-supported clades, corresponding to the traditionally circumscribed subgenera Eumitria, Dolichousnea, Neuropogon and Usnea. However, characters that have been used to describe these clades are often homoplasious within the phylogeny and their parallel evolution is suggested. On the other hand, most of the species were reconstructed as monophyletic, indicating that combinations of phenotypic characters are suitable discriminators for delimitating species, but are inadequate to describe generic subdivisions.

Transient colocalization of X-inactivation centres accompanies the initiation of X inactivation.

The initial differential treatment of the two X chromosomes during X-chromosome inactivation is controlled by the X-inactivation centre (Xic). This locus determines how many X chromosomes are present in a cell ('counting') and which X chromosome will be inactivated in female cells ('choice'). Critical control sequences in the Xic include the non-coding RNAs Xist and Tsix, and long-range chromatin elements. However, little is known about the process that ensures that X inactivation is triggered appropriately when more than one Xic is present in a cell. Using three-dimensional fluorescence in situ hybridization (FISH) analysis, we showed that the two Xics transiently colocalize, just before X inactivation, in differentiating female embryonic stem cells. Using Xic transgenes capable of imprinted but not random X inactivation, and Xic deletions that disrupt random X inactivation, we demonstrated that Xic colocalization is linked to Xic function in random X inactivation. Both long-range sequences and the Tsix element, which generates the antisense transcript to Xist, are required for the transient interaction of Xics. We propose that transient colocalization of Xics may be necessary for a cell to determine Xic number and to ensure the correct initiation of X inactivation.

2-D structure of the A region of Xist RNA and its implication for PRC2 association.

In placental mammals, inactivation of one of the X chromosomes in female cells ensures sex chromosome dosage compensation. The 17 kb non-coding Xist RNA is crucial to this process and accumulates on the future inactive X chromosome. The most conserved Xist RNA region, the A region, contains eight or nine repeats separated by U-rich spacers. It is implicated in the recruitment of late inactivated X genes to the silencing compartment and likely in the recruitment of complex PRC2. Little is known about the structure of the A region and more generally about Xist RNA structure. Knowledge of its structure is restricted to an NMR study of a single A repeat element. Our study is the first experimental analysis of the structure of the entire A region in solution. By the use of chemical and enzymatic probes and FRET experiments, using oligonucleotides carrying fluorescent dyes, we resolved problems linked to sequence redundancies and established a 2-D structure for the A region that contains two long stem-loop structures each including four repeats. Interactions formed between repeats and between repeats and spacers stabilize these structures. Conservation of the spacer terminal sequences allows formation of such structures in all sequenced Xist RNAs. By combination of RNP affinity chromatography, immunoprecipitation assays, mass spectrometry, and Western blot analysis, we demonstrate that the A region can associate with components of the PRC2 complex in mouse ES cell nuclear extracts. Whilst a single four-repeat motif is able to associate with components of this complex, recruitment of Suz12 is clearly more efficient when the entire A region is present. Our data with their emphasis on the importance of inter-repeat pairing change fundamentally our conception of the 2-D structure of the A region of Xist RNA and support its possible implication in recruitment of the PRC2 complex.

Thiadiazoles as new inhibitors of diacylglycerol acyltransferase type 1.

A novel class of DGAT1 inhibitors containing a thiadiazole core has been discovered. Chemical optimization lead to inhibitors of human DGAT1 with an appropriate ADME profile and that show in vivo activity in target tissues.

Random X-chromosome inactivation: skewing lessons for mice and men.

The mammalian X-chromosome exists in two flavors, active and inactive, in each cell of the adult female. This phenomenon originates from the process of random choice occurring early in development in a small number of progenitor cells in which the decision is made to inactivate either one or the other X chromosome on a cell-autonomous basis. Once made, this initial decision is irreversible, although exceptions exist in specific chromosomal territories and cell lineages. Recent findings implicate various factors, including non-coding RNAs and chromatin modification complexes, as effectors in the initiation and maintenance of X-chromosome inactivation. The functional redundancy of such factors almost certainly plays an important role in the stability of the inactive X. Studying skewing or bias opens an important opportunity for understanding facets of the random choice process.

Phylogeny of the subgenus Eumitria in Tanzania.

Several Usnea species in subgenus Eumitria (Parmeliaceae, lichenized Ascomycota) have been described from East Africa in the past decades. These have been based on morphology and chemistry data while molecular studies remain very limited. In this paper we are for the first time publishing phylogenetic analyses along with morphological and chemical data for Eumitria. ‬A total of 62 new sequences of Eumitria (26 ITS, 20 nuLSU, 6 MCM7, 10 RPB1) were generated in this study. nuLSU, MCM7 and RPB1 sequences are here for the first time reported for U. baileyi. A phylogeny of subgenus Eumitria from Tanzania based on Bayesian and maximum likelihood analyses of a concatenated four-loci data set is presented, confirming the monophyly of Eumitria. Further, secondary chemistry and variation in characters, such as the pigmentation of the central axis and branch shape were investigated.

The molecular logic of Nanog-induced self-renewal in mouse embryonic stem cells.

Transcription factor networks, together with histone modifications and signalling pathways, underlie the establishment and maintenance of gene regulatory architectures associated with the molecular identity of each cell type. However, how master transcription factors individually impact the epigenomic landscape and orchestrate the behaviour of regulatory networks under different environmental constraints is only partially understood. Here, we show that the transcription factor Nanog deploys multiple distinct mechanisms to enhance embryonic stem cell self-renewal. In the presence of LIF, which fosters self-renewal, Nanog rewires the pluripotency network by promoting chromatin accessibility and binding of other pluripotency factors to thousands of enhancers. In the absence of LIF, Nanog blocks differentiation by sustaining H3K27me3, a repressive histone mark, at developmental regulators. Among those, we show that the repression of Otx2 plays a preponderant role. Our results underscore the versatility of master transcription factors, such as Nanog, to globally influence gene regulation during developmental processes.

Ecophysiology and phylogeny of new terricolous and epiphytic chlorolichens in a fog oasis of the Atacama Desert.

The Atacama Desert is one of the driest and probably oldest deserts on Earth where only a few extremophile organisms are able to survive. This study investigated two terricolous and two epiphytic lichens from the fog oasis "Las Lomitas" within the National Park Pan de Azúcar which represents a refugium for a few vascular desert plants and many lichens that can thrive on fog and dew alone. Ecophysiological measurements and climate records were combined with molecular data of the mycobiont, their green algal photobionts and lichenicolous fungi to gain information about the ecology of lichens within the fog oasis. Phylogenetic and morphological investigations led to the identification and description of the new lichen species Acarospora conafii sp. nov. as well as the lichenicolous fungi that accompanied them and revealed the trebouxioid character of all lichen photobionts. Their photosynthetic responses were compared during natural scenarios such as reactivation by high air humidity and in situ fog events to elucidate the activation strategies of this lichen community. Epiphytic lichens showed photosynthetic activity that was rapidly induced by fog and high relative air humidity whereas terricolous lichens were only activated by fog.

Overcoming deterrent metabolites by gaining essential nutrients: A lichen/snail case study.

Specialised metabolites in lichens are generally considered repellent compounds by consumers. Nevertheless, if the only food available is lichens rich in specialised metabolites, lichenophages must implement strategies to overcome the toxicity of these metabolites. Thus, the balance between phagostimulant nutrients and deterrent metabolites could play a key role in feeding preferences. To further understand lichen-gastropod interactions, we studied the feeding behaviour and consumption in Notodiscus hookeri, the land snail native to sub-Antarctic islands. The lichen Usnea taylorii was used because of its simple chemistry, its richness in usnic acid (specialised metabolite) and arabitol (primary metabolite) and its presence in snail habitats. Choice tests in arenas with intact lichens versus acetone-rinsed lichens were carried out to study the influence of specialised metabolites on snail behaviour and feeding preference. Simultaneously, usnic acid and arabitol were quantified and located within the lichen thallus using HPLC-DAD-MS and in situ imaging by mass spectrometry to assess whether their spatial distribution explained preferential snail grazing. No-choice feeding experiments, with the pure metabolites embedded in an artificial diet, defined a gradual gustatory response, from strong repellence (usnic acid) to high appetence (D-arabitol). This case study demonstrates that the nutritional activity of N. hookeri is governed by the chemical quality of the food and primarily by nutrient availability (arabitol), despite the presence of deterrent metabolite (usnic acid).

The lichens of the Alps - an annotated checklist.

This is the first attempt to provide an overview of the lichen diversity of the Alps, one of the biogegraphically most important and emblematic mountain systems worldwide. The checklist includes all lichenised species, plus a set of non- or doubtfully lichenised taxa frequently treated by lichenologists, excluding non-lichenised lichenicolous fungi. Largely based on recent national or regional checklists, it provides a list of all infrageneric taxa (with synonyms) hitherto reported from the Alps, with data on their distribution in eight countries (Austria, France, Germany, Liechtenstein, Monaco, Italy, Slovenia, Switzerland) and in 42 Operational Geographic Units, mostly corresponding to administrative subdivisions within the countries. Data on the main substrates and on the altitudinal distribution are also provided. A short note points to the main ecological requirements of each taxon and/or to open taxonomic problems. Particularly poorly known taxa are flagged and often provided with a short description, to attract the attention of specialists. The total number of infrageneric taxa is 3,163, including 117 non- or doubtfully lichenised taxa. The richness of the lichen biota fairly well corresponds with the percent of the Alpine area occupied by each country: Austria (2,337 taxa), Italy (2,169), France (2,028), Switzerland (1,835), Germany (1,168), Slovenia (890) and Lichtenstein (152), no lichen having ever been reported from Monaco. The number of poorly known taxa is quite high (604, 19.1% of the total), which indicates that, in spite of the Alps being one of the lichenologically most studied mountain systems worldwide, much work is still needed to reach a satisfactory picture of their real lichen diversity. Thirteen new combinations are proposed in the genera Agonimia, Aspicilia, Bagliettoa, Bellemerea, Carbonea, Lepra, Miriquidica, Polysporina, Protothelenella, Pseudosagedia and Thelidium.

Functional assessment of mitral regurgitation by transthoracic echocardiography using standardized imaging planes diagnostic accuracy and outcome implications.

OBJECTIVES: We sought to assess the value of transthoracic echocardiography (TTE) using standardized imaging planes for the functional analysis of mitral regurgitation (MR) as well as for postoperative outcome implications. BACKGROUND: The feasibility of mitral valve repair is based on functional assessment of MR, mainly by transesophageal echocardiography (TEE). Considering the recent advances in TTE imaging, the incremental value of TEE in this setting needs to be re-examined. METHODS: Consecutive patients (n = 279; 181 men; median age 68 years [quartiles, 61 to 74]) who underwent surgery for MR were enrolled prospectively in two tertiary care centers. The accuracy of TTE (harmonic imaging) versus TEE for functional assessment of MR was evaluated against surgical findings. RESULTS: Valve repair (n = 237 patients, 85%) or replacement (n = 42) was predicted accurately by TTE in 97% of cases; TEE added significant information for only two patients. In the subgroup of degenerative MR (n = 190), agreement with surgical findings for the localization of prolapsed segments was 91% for TTE (kappa, 0.81) and 93% for TEE (kappa, 0.85) without incremental value of TEE (p = 0.40). Patients with single prolapse of the middle posterior scallop (P2) had a better postoperative outcome as compared with patients who had non-P2 lesions (p = 0.008). Furthermore, mitral replacement predicted by TTE was an independent predictor for postoperative long-term mortality (odds ratio 5.7, 95% confidence interval 1.97 to 16.4, p = 0.001). CONCLUSIONS: In experienced hands, functional assessment of MR by TTE can predict accurately valve repairability and has a strong influence on postoperative outcome. Thus, in most cases preoperative TEE is not mandatory, provided intraoperative TEE is performed.

Live cell imaging of the nascent inactive X chromosome during the early differentiation process of naive ES cells towards epiblast stem cells.

Random X-chromosome inactivation ensures dosage compensation in mammals through the transcriptional silencing of one of the two X chromosomes present in each female cell. Silencing is initiated in the differentiating epiblast of the mouse female embryos through coating of the nascent inactive X chromosome by the non-coding RNA Xist, which subsequently recruits the Polycomb Complex PRC2 leading to histone H3-K27 methylation. Here we examined in mouse ES cells the early steps of the transition from naive ES cells towards epiblast stem cells as a model for inducing X chromosome inactivation in vitro. We show that these conditions efficiently induce random XCI. Importantly, in a transient phase of this differentiation pathway, both X chromosomes are coated with Xist RNA in up to 15% of the XX cells. In an attempt to determine the dynamics of this process, we designed a strategy aimed at visualizing the nascent inactive X-chromosome in live cells. We generated transgenic female XX ES cells expressing the PRC2 component Ezh2 fused to the fluorescent protein Venus. The fluorescent fusion protein was expressed at sub-physiological levels and located in nuclei of ES cells. Upon differentiation of ES cell towards epiblast stem cell fate, Venus-fluorescent territories appearing in interphase nuclei were identified as nascent inactive X chromosomes by their association with Xist RNA. Imaging of Ezh2-Venus for up to 24 hours during the differentiation process showed survival of some cells with two fluorescent domains and a surprising dynamics of the fluorescent territories across cell division and in the course of the differentiation process. Our data reveal a strategy for visualizing the nascent inactive X chromosome and suggests the possibility for a large plasticity of the nascent inactive X chromosome.

New lessons from random X-chromosome inactivation in the mouse.

X-chromosome inactivation (XCI) ensures dosage compensation in mammals. Random XCI is a process where a single X chromosome is silenced in each cell of the epiblast of mouse female embryos. Operating at the level of an entire chromosome, XCI is a major paradigm for epigenetic processes. Here we review the most recent discoveries concerning the role of long noncoding RNAs, pluripotency factors, and chromosome structure in random XCI.

A role for non-coding Tsix transcription in partitioning chromatin domains within the mouse X-inactivation centre.

BACKGROUND: Delimiting distinct chromatin domains is essential for temporal and spatial regulation of gene expression. Within the X-inactivation centre region (Xic), the Xist locus, which triggers X-inactivation, is juxtaposed to a large domain of H3K27 trimethylation (H3K27me3). RESULTS: We describe here that developmentally regulated transcription of Tsix, a crucial non-coding antisense to Xist, is required to block the spreading of the H3K27me3 domain to the adjacent H3K4me2-rich Xist region. Analyses of a series of distinct Tsix mutations suggest that the underlying mechanism involves the RNA Polymerase II accumulating at the Tsix 3'-end. Furthermore, we report additional unexpected long-range effects of Tsix on the distal sub-region of the Xic, involved in Xic-Xic trans-interactions. CONCLUSION: These data point toward a role for transcription of non-coding RNAs as a developmental strategy for the establishment of functionally distinct domains within the mammalian genome.

An essential role for the DXPas34 tandem repeat and Tsix transcription in the counting process of X chromosome inactivation.

A counting process senses the X chromosome/autosome ratio and ensures that X chromosome inactivation (XCI) initiates in the early female (XX) embryo and in differentiating female ES cells but not in their male (XY) counterparts. Counting depends on the X inactivation center (Xic), which contains the Xist gene encoding a nuclear RNA, which coats the inactive X chromosome and induces gene silencing. A 37-kb sequence lying 3' to the Xist gene is known to prevent initiation of XCI in male differentiating ES cells. This region contains the major and minor promoters of the Tsix gene, which runs antisense to Xist, and the DXPas34 tandem repeat lying close to the Tsix major promoter. We have addressed the role of these elements in counting by using male ES cells. Targeted deletion of DXPas34 impaired recruitment of RNA-polymerase II and TFIIB at the Tsix major promoter, resulting in low levels of Tsix expression in ES cells and moderate ectopic initiation of XCI upon differentiation. A deletion extending 3' to Xist and including the Tsix major promoter resulted in almost complete impairment of Tsix transcription and in efficient ectopic XCI upon differentiation of male ES cells. Internal deletions within the Tsix gene did not affect significantly the level of antisense transcription within Xist and had only minor effects upon differentiation. Our results identify a function for DXPas34 in murine XCI and demonstrate the critical role of Tsix transcription in preventing XCI in differentiating male ES cells and in normal functioning of the counting pathway.