A Uniform System for the Annotation of Vertebrate microRNA Genes and the Evolution of the Human microRNAome.

Although microRNAs (miRNAs) are among the most intensively studied molecules of the past 20 years, determining what is and what is not a miRNA has not been straightforward. Here, we present a uniform system for the annotation and nomenclature of miRNA genes. We show that less than a third of the 1,881 human miRBase entries, and only approximately 16% of the 7,095 metazoan miRBase entries, are robustly supported as miRNA genes. Furthermore, we show that the human repertoire of miRNAs has been shaped by periods of intense miRNA innovation and that mature gene products show a very different tempo and mode of sequence evolution than star products. We establish a new open access database--MirGeneDB ( http://mirgenedb.org )--to catalog this set of miRNAs, which complements the efforts of miRBase but differs from it by annotating the mature versus star products and by imposing an evolutionary hierarchy upon this curated and consistently named repertoire.

MicroRNA annotation of plant genomes - Do it right or not at all.

MicroRNAs are non-coding regulators of gene expression and key factors in development, disease, and targets for bioengineering. Consequently, microRNAs have become essential elements of already burgeoning draft plant genome descriptions where their annotation is often particularly poor, contributing unduly to the corruption of public databases. Using the Citrus sinensis as an example, we highlight and review common failings of miRNAome annotations. Understanding and exploiting the role of miRNAs in plant biology will be stymied unless the research community acts decisively to improve the accuracy of miRNAome annotations. We encourage genome annotation teams to do it right or not at all.

Molecular signatures of plastic phenotypes in two eusocial insect species with simple societies.

Phenotypic plasticity is important in adaptation and shapes the evolution of organisms. However, we understand little about what aspects of the genome are important in facilitating plasticity. Eusocial insect societies produce plastic phenotypes from the same genome, as reproductives (queens) and nonreproductives (workers). The greatest plasticity is found in the simple eusocial insect societies in which individuals retain the ability to switch between reproductive and nonreproductive phenotypes as adults. We lack comprehensive data on the molecular basis of plastic phenotypes. Here, we sequenced genomes, microRNAs (miRNAs), and multiple transcriptomes and methylomes from individual brains in a wasp (Polistes canadensis) and an ant (Dinoponera quadriceps) that live in simple eusocial societies. In both species, we found few differences between phenotypes at the transcriptional level, with little functional specialization, and no evidence that phenotype-specific gene expression is driven by DNA methylation or miRNAs. Instead, phenotypic differentiation was defined more subtly by nonrandom transcriptional network organization, with roles in these networks for both conserved and taxon-restricted genes. The general lack of highly methylated regions or methylome patterning in both species may be an important mechanism for achieving plasticity among phenotypes during adulthood. These findings define previously unidentified hypotheses on the genomic processes that facilitate plasticity and suggest that the molecular hallmarks of social behavior are likely to differ with the level of social complexity.

Uncertain-tree: discriminating among competing approaches to the phylogenetic analysis of phenotype data.

Morphological data provide the only means of classifying the majority of life's history, but the choice between competing phylogenetic methods for the analysis of morphology is unclear. Traditionally, parsimony methods have been favoured but recent studies have shown that these approaches are less accurate than the Bayesian implementation of the Mk model. Here we expand on these findings in several ways: we assess the impact of tree shape and maximum-likelihood estimation using the Mk model, as well as analysing data composed of both binary and multistate characters. We find that all methods struggle to correctly resolve deep clades within asymmetric trees, and when analysing small character matrices. The Bayesian Mk model is the most accurate method for estimating topology, but with lower resolution than other methods. Equal weights parsimony is more accurate than implied weights parsimony, and maximum-likelihood estimation using the Mk model is the least accurate method. We conclude that the Bayesian implementation of the Mk model should be the default method for phylogenetic estimation from phenotype datasets, and we explore the implications of our simulations in reanalysing several empirical morphological character matrices. A consequence of our finding is that high levels of resolution or the ability to classify species or groups with much confidence should not be expected when using small datasets. It is now necessary to depart from the traditional parsimony paradigms of constructing character matrices, towards datasets constructed explicitly for Bayesian methods.

microRNAs and the evolution of complex multicellularity: identification of a large, diverse complement of microRNAs in the brown alga Ectocarpus.

There is currently convincing evidence that microRNAs have evolved independently in at least six different eukaryotic lineages: animals, land plants, chlorophyte green algae, demosponges, slime molds and brown algae. MicroRNAs from different lineages are not homologous but some structural features are strongly conserved across the eukaryotic tree allowing the application of stringent criteria to identify novel microRNA loci. A large set of 63 microRNA families was identified in the brown alga Ectocarpus based on mapping of RNA-seq data and nine microRNAs were confirmed by northern blotting. The Ectocarpus microRNAs are highly diverse at the sequence level with few multi-gene families, and do not tend to occur in clusters but exhibit some highly conserved structural features such as the presence of a uracil at the first residue. No homologues of Ectocarpus microRNAs were found in other stramenopile genomes indicating that they emerged late in stramenopile evolution and are perhaps specific to the brown algae. The large number of microRNA loci in Ectocarpus is consistent with the developmental complexity of many brown algal species and supports a proposed link between the emergence and expansion of microRNA regulatory systems and the evolution of complex multicellularity.

Bayesian methods outperform parsimony but at the expense of precision in the estimation of phylogeny from discrete morphological data.

Different analytical methods can yield competing interpretations of evolutionary history and, currently, there is no definitive method for phylogenetic reconstruction using morphological data. Parsimony has been the primary method for analysing morphological data, but there has been a resurgence of interest in the likelihood-based Mk-model. Here, we test the performance of the Bayesian implementation of the Mk-model relative to both equal and implied-weight implementations of parsimony. Using simulated morphological data, we demonstrate that the Mk-model outperforms equal-weights parsimony in terms of topological accuracy, and implied-weights performs the most poorly. However, the Mk-model produces phylogenies that have less resolution than parsimony methods. This difference in the accuracy and precision of parsimony and Bayesian approaches to topology estimation needs to be considered when selecting a method for phylogeny reconstruction.

Stimulation of cortical bone formation with thienopyrimidine based inhibitors of Notum Pectinacetylesterase.

A group of small molecule thienopyrimidine inhibitors of Notum Pectinacetylesterase are described. We explored both 2-((5,6-thieno[2,3-d]pyrimidin-4-yl)thio)acetic acids and 2-((6,7-thieno[3,2-d]pyrimidin-4-yl)thio)acetic acids. In both series, highly potent, orally active Notum Pectinacetylesterase inhibitors were identified.

4H-Thieno[3,2-c]chromene based inhibitors of Notum Pectinacetylesterase.

A group of small molecule thienochromenes inhibitors of Notum Pectinacetylesterase are described. We developed SAR on three series based on carbon, oxygen and sulfur replacement of the 5-position. In each series, highly potent Notum Pectinacetylesterase inhibitors were identified.

miRNAs: small genes with big potential in metazoan phylogenetics.

microRNAs (miRNAs) are a key component of gene regulatory networks and have been implicated in the regulation of virtually every biological process found in multicellular eukaryotes. What makes them interesting from a phylogenetic perspective is the high conservation of primary sequence between taxa, their accrual in metazoan genomes through evolutionary time, and the rarity of secondary loss in most metazoan taxa. Despite these properties, the use of miRNAs as phylogenetic markers has not yet been discussed within a clear conceptual framework. Here we highlight five properties of miRNAs that underlie their utility in phylogenetics: 1) The processes of miRNA biogenesis enable the identification of novel miRNAs without prior knowledge of sequence; 2) The continuous addition of miRNA families to metazoan genomes through evolutionary time; 3) The low level of secondary gene loss in most metazoan taxa; 4) The low substitution rate in the mature miRNA sequence; and 5) The small probability of convergent evolution of two miRNAs. Phylogenetic analyses using both Bayesian and parsimony methods on a eumetazoan miRNA data set highlight the potential of miRNAs to become an invaluable new tool, especially when used as an additional line of evidence, to resolve previously intractable nodes within the tree of life.

Do miRNAs have a deep evolutionary history?

The recent discovery of microRNAs (miRNAs) in unicellular eukaryotes, including miRNAs known previously only from animals or plants, implies that miRNAs have a deep evolutionary history among eukaryotes. This contrasts with the prevailing view that miRNAs evolved convergently in animals and plants. We re-evaluate the evidence and find that none of the 73 plant and animal miRNAs described from protists meet the required criteria for miRNA annotation and, by implication, animals and plants did not acquire any of their respective miRNA genes from the crown ancestor of eukaryotes. Furthermore, of the 159 novel miRNAs previously identified among the seven species of unicellular protists examined, only 28 from the algae Ectocarpus and Chlamydomonas, meet the criteria for miRNA annotation. Therefore, at present only five groups of eukaryotes are known to possess miRNAs, indicating that miRNAs have evolved independently within eukaryotes through exaptation of their shared inherited RNAi machinery.

Inhaled treprostinil in patients with pulmonary hypertension associated with interstitial lung disease with less severe haemodynamics: a post hoc analysis of the INCREASE study.

BACKGROUND: Inhaled treprostinil (iTre) is the only treatment approved for pulmonary hypertension due to interstitial lung disease (PH-ILD) to improve exercise capacity. This post hoc analysis evaluated clinical worsening and PH-ILD exacerbations from the 16-week INCREASE study and change in 6-minute walking distance (6MWD) in the INCREASE open-label extension (OLE) in patients with less severe haemodynamics. METHODS: Patients were stratified by baseline pulmonary vascular resistance (PVR) of <4 Wood units (WU) versus ≥4 WU and <5 WU versus ≥5 WU. Exacerbations of underlying lung disease, clinical worsening and change in N-terminal prohormone of brain natriuretic peptide (NT-proBNP) in INCREASE were evaluated. For the OLE, patients previously assigned to placebo were considered to have a 16-week treatment delay. 6MWD and clinical events in the OLE were evaluated by PVR subgroup. RESULTS: Of the 326 patients enrolled in INCREASE, patients with less severe haemodynamics receiving iTre had fewer exacerbations of underlying lung disease and clinical worsening events. This was supported by the Bayesian analysis of the risk of disease progression (HR<1), and significant decreases in NT-proBNP levels. In the OLE, patients without a treatment delay had improved exercise capacity after 1-year compared with those with a 16-week treatment delay (22.1 m vs -10.3 m). Patients with a PVR of ≤5 WU without a treatment delay had a change of 5.5 m compared with -8.2 m for those with a treatment delay. Patients without a treatment delay had a prolonged time to hospitalisation, lung disease exacerbation and death. CONCLUSION: Treatment with iTre led to consistent benefits in clinical outcomes in patients with PH-ILD and less severe haemodynamics. Earlier treatment in less severe PH-ILD may lead to better exercise capacity long-term, however, the subgroup analyses in this post hoc study were underpowered and confirmation of these findings is needed.

Reducing rates of readmission and development of an outpatient management plan in pulmonary hypertension: lessons from congestive heart failure management.

Pulmonary hypertension currently has minimal guidelines for outpatient disease management. Congestive heart failure studies, however, have shown effectiveness of disease management plans in reducing all-cause mortality and all-cause and congestive heart failure-related hospital readmissions. Heart failure exacerbation is a common reason for readmission in both pulmonary hypertension and congestive heart failure. Our aim was to review individual studies and comprehensive meta-analyses to identify effective congestive heart failure interventions that can be used to develop similar disease management plans for pulmonary hypertension. A comprehensive literature review from 1993 to 2019 included original articles, systematic reviews, and meta-analyses. We reviewed topics of outpatient congestive heart failure interventions to decrease congestive heart failure mortality and readmission and patient management strategies in congestive heart failure. The most studied interventions included case management, multidisciplinary intervention, structured telephone strategy, and tele-monitoring. Case management showed decreased all-cause mortality at 12 months, all-cause readmission at 12 months, and congestive heart failure readmission at 6 and 12 months. Multidisciplinary intervention resulted in decreased all-cause readmission and congestive heart failure readmission. There was some discrepancy on effectiveness of tele-monitoring programs in individual studies; however, meta-analyses suggest tele-monitoring provided reduced all-cause mortality and risk of congestive heart failure hospitalization. Structured telephone strategy had similar results to tele-monitoring including decreased risk of congestive heart failure hospitalization, without effect on mortality. Extrapolating from congestive heart failure data, it seems strategies to improve the health of pulmonary hypertension patients and development of comprehensive care programs should include structured telephone strategy and/or tele-monitoring, case management strategies, and multidisciplinary interventions.

5-Fluorocytosine derivatives as inhibitors of deoxycytidine kinase.

A series of potent piperidine-linked cytosine derivatives were prepared as inhibitors of deoxycytidine kinase (dCK). Compound 9h was discovered to be a potent inhibitor of dCK and shows a good combination of cellular potency and pharmacokinetic parameters. Compound 9h blocks the incorporation of radiolabeled cytosine into mouse T-cells in vitro, as well as in vivo in mice following a T-cell challenge.

Lead optimization and structure-based design of potent and bioavailable deoxycytidine kinase inhibitors.

A series of deoxycytidine kinase inhibitors was simultaneously optimized for potency and PK properties. A co-crystal structure then allowed merging this series with a high throughput screening hit to afford a highly potent, selective and orally bioavailable inhibitor, compound 10. This compound showed dose dependent inhibition of deoxycytidine kinase in vivo.

Long-term results of the DelIVery for Pulmonary Arterial Hypertension trial.

BACKGROUND: The DelIVery for Pulmonary Arterial Hypertension clinical trial was a multi-center, prospective, single arm, Investigational Device Exemption study utilizing a fully implantable, programmable intravascular delivery system consisting of a pump and a catheter for intravenous treprostinil. The study met its primary endpoint and demonstrated that the intravascular delivery system significantly reduced catheter related complications at 22,000 subject-days of follow-up compared with a predefined objective performance criterion. Here we summarize the results obtained during a 6.4-year follow-up period. METHODS: Throughout study follow-up, participants had clinic visits and medication refills at least every 12 weeks (dependent on the subjects' dose). All adverse events and intravascular delivery system complications were evaluated and recorded. RESULTS: Sixty pulmonary arterial hypertension subjects were followed post device implantation for approximately 282 patient-years (range 87 days to 6.4 years). Of the 60 subjects, 14 died (1 related to intravascular delivery system pump failure), 2 withdrew after lung transplants, and 2 withdrew due to pump pocket infection. No catheter-related bloodstream infections, catheter thrombosis or occlusions, or catheter kinks occurred through 282 patient-years. Two participants had adverse events of abdominal pain, rash, due to subcutaneous treprostinil "leaks" after one catheter puncture and one catheter laceration during pump refill and replacement, respectively. Eight pump failure events occurred: seven pump motor stalls and one early replacement (faulty battery). CONCLUSION: Delivery of treprostinil with an intravascular delivery system is a safe alternative to an external delivery system, while providing enhanced life experiences. To preserve the risk-benefit ratio, treatment at specialized pulmonary arterial hypertension centers is recommended until training is disseminated at other sites.

NOTUM inhibition increases endocortical bone formation and bone strength.

The disability, mortality and costs caused by non-vertebral osteoporotic fractures are enormous. Existing osteoporosis therapies are highly effective at reducing vertebral but not non-vertebral fractures. Cortical bone is a major determinant of non-vertebral bone strength. To identify novel osteoporosis drug targets, we phenotyped cortical bone of 3 366 viable mouse strains with global knockouts of druggable genes. Cortical bone thickness was substantially elevated in Notum -/- mice. NOTUM is a secreted WNT lipase and we observed high NOTUM expression in cortical bone and osteoblasts but not osteoclasts. Three orally active small molecules and a neutralizing antibody inhibiting NOTUM lipase activity were developed. They increased cortical bone thickness and strength at multiple skeletal sites in both gonadal intact and ovariectomized rodents by stimulating endocortical bone formation. Thus, inhibition of NOTUM activity is a potential novel anabolic therapy for strengthening cortical bone and preventing non-vertebral fractures.

Dinosaurs and the Cretaceous Terrestrial Revolution.

The observed diversity of dinosaurs reached its highest peak during the mid- and Late Cretaceous, the 50 Myr that preceded their extinction, and yet this explosion of dinosaur diversity may be explained largely by sampling bias. It has long been debated whether dinosaurs were part of the Cretaceous Terrestrial Revolution (KTR), from 125-80 Myr ago, when flowering plants, herbivorous and social insects, squamates, birds and mammals all underwent a rapid expansion. Although an apparent explosion of dinosaur diversity occurred in the mid-Cretaceous, coinciding with the emergence of new groups (e.g. neoceratopsians, ankylosaurid ankylosaurs, hadrosaurids and pachycephalosaurs), results from the first quantitative study of diversification applied to a new supertree of dinosaurs show that this apparent burst in dinosaurian diversity in the last 18 Myr of the Cretaceous is a sampling artefact. Indeed, major diversification shifts occurred largely in the first one-third of the group's history. Despite the appearance of new clades of medium to large herbivores and carnivores later in dinosaur history, these new originations do not correspond to significant diversification shifts. Instead, the overall geometry of the Cretaceous part of the dinosaur tree does not depart from the null hypothesis of an equal rates model of lineage branching. Furthermore, we conclude that dinosaurs did not experience a progressive decline at the end of the Cretaceous, nor was their evolution driven directly by the KTR.

Well-Annotated microRNAomes Do Not Evidence Pervasive miRNA Loss.

microRNAs are conserved noncoding regulatory factors implicated in diverse physiological and developmental processes in multicellular organisms, as causal macroevolutionary agents and for phylogeny inference. However, the conservation and phylogenetic utility of microRNAs has been questioned on evidence of pervasive loss. Here, we show that apparent widespread losses are, largely, an artefact of poorly sampled and annotated microRNAomes. Using a curated data set of animal microRNAomes, we reject the view that miRNA families are never lost, but they are rarely lost (92% are never lost). A small number of families account for a majority of losses (1.7% of families account for >45% losses), and losses are associated with lineages exhibiting phenotypic simplification. Phylogenetic analyses based on the presence/absence of microRNA families among animal lineages, and based on microRNA sequences among Osteichthyes, demonstrate the power of these small data sets in phylogenetic inference. Perceptions of widespread evolutionary loss of microRNA families are due to the uncritical use of public archives corrupted by spurious microRNA annotations, and failure to discriminate false absences that occur because of incomplete microRNAome annotation.