Ribosome subunit attrition and activation of the p53-MDM4 axis dominate the response of MLL-rearranged cancer cells to WDR5 WIN site inhibition.

The chromatin-associated protein WD Repeat Domain 5 (WDR5) is a promising target for cancer drug discovery, with most efforts blocking an arginine-binding cavity on the protein called the 'WIN' site that tethers WDR5 to chromatin. WIN site inhibitors (WINi) are active against multiple cancer cell types in vitro, the most notable of which are those derived from MLL-rearranged (MLLr) leukemias. Peptidomimetic WINi were originally proposed to inhibit MLLr cells via dysregulation of genes connected to hematopoietic stem cell expansion. Our discovery and interrogation of small-molecule WINi, however, revealed that they act in MLLr cell lines to suppress ribosome protein gene (RPG) transcription, induce nucleolar stress, and activate p53. Because there is no precedent for an anticancer strategy that specifically targets RPG expression, we took an integrated multi-omics approach to further interrogate the mechanism of action of WINi in human MLLr cancer cells. We show that WINi induce depletion of the stock of ribosomes, accompanied by a broad yet modest translational choke and changes in alternative mRNA splicing that inactivate the p53 antagonist MDM4. We also show that WINi are synergistic with agents including venetoclax and BET-bromodomain inhibitors. Together, these studies reinforce the concept that WINi are a novel type of ribosome-directed anticancer therapy and provide a resource to support their clinical implementation in MLLr leukemias and other malignancies.

Ribosome subunit attrition and activation of the p53-MDM4 axis dominate the response of MLL-rearranged cancer cells to WDR5 WIN site inhibition.

The chromatin-associated protein WD Repeat Domain 5 (WDR5) is a promising target for cancer drug discovery, with most efforts blocking an arginine-binding cavity on the protein called the "WIN" site that tethers WDR5 to chromatin. WIN site inhibitors (WINi) are active against multiple cancer cell types in vitro, the most notable of which are those derived from MLL-rearranged (MLLr) leukemias. Peptidomimetic WINi were originally proposed to inhibit MLLr cells via dysregulation of genes connected to hematopoietic stem cell expansion. Our discovery and interrogation of small molecule WIN site inhibitors, however, revealed that they act in MLLr cell lines to suppress ribosome protein gene (RPG) transcription, induce nucleolar stress, and activate p53. Because there is no precedent for an anti-cancer strategy that specifically targets RPG expression, we took an integrated multi-omics approach to further interrogate the mechanism of action of WINi in MLLr cancer cells. We show that WINi induce depletion of the stock of ribosomes, accompanied by a broad yet modest translational choke and changes in alternative mRNA splicing that inactivate the p53 antagonist MDM4. We also show that WINi are synergistic with agents including venetoclax and BET-bromodomain inhibitors. Together, these studies reinforce the concept that WINi are a novel type of ribosome-directed anti-cancer therapy and provide a resource to support their clinical implementation in MLLr leukemias and other malignancies.

Synergistic action of WDR5 and HDM2 inhibitors in SMARCB1-deficient cancer cells.

Rhabdoid tumors (RT) are rare and deadly pediatric cancers driven by loss of SMARCB1, which encodes the SNF5 component of the SWI/SNF chromatin remodeler. Loss of SMARCB1 is associated with a complex set of phenotypic changes including vulnerability to inhibitors of protein synthesis and of the p53 ubiquitin-ligase HDM2. Recently, we discovered small molecule inhibitors of the 'WIN' site of WDR5, which in MLL-rearranged leukemia cells decrease the expression of a set of genes linked to protein synthesis, inducing a translational choke and causing p53-dependent inhibition of proliferation. Here, we characterize how WIN site inhibitors act in RT cells. As in leukemia cells, WIN site inhibition in RT cells causes the comprehensive displacement of WDR5 from chromatin, resulting in a decrease in protein synthesis gene expression. Unlike leukemia cells, however, the growth response of RT cells to WIN site blockade is independent of p53. Exploiting this observation, we demonstrate that WIN site inhibitor synergizes with an HDM2 antagonist to induce p53 and block RT cell proliferation in vitro. These data reveal a p53-independent action of WIN site inhibitors and forecast that future strategies to treat RT could be based on dual WDR5/HDM2 inhibition.

[Allergic Broncho-Pulmonary Aspergillosis (ABPA) in cystic fibrosis: Mechanisms, diagnosis and therapeutic options]. / Aspergillose bronchopulmonaire allergique (ABPA) et mucoviscidose : mécanismes, diagnostic et alternatives thérapeutiques.

INTRODUCTION: Fungal aspergillosis colonization and allergic bronchopulmonary aspergillosis (ABPA) can have a strong impact on the prognosis in cystic fibrosis (CF). We conducted round table discussions involving French experts from pediatric and adult centers caring for patients with CF, microbiologists, radiologists and pharmacists. The aim was to explore the current state of knowledge on: the pathophysiological mechanisms of Aspergillus and other micromycetes infections in CF (such as Scedosporium sp.), and on the clinico-biological diagnosis of ABPA. In perspective, the experts explored the role of imaging in the diagnosis of APBA, specifically CT and MRI; as well as the role of bronchoscopy in the management. We also reviewed the therapeutic management, including different corticosteroid regimens, antifungals and anti-IgE antibodies. CONCLUSION: The diagnosis of ABPA in CF should be based on more standardized biological assays and imaging to optimize treatment and follow-up.

The demise of a wonder: Evolutionary history and conservation assessments of the Wonder Gecko Teratoscincus keyserlingii (Gekkota, Sphaerodactylidae) in Arabia.

Effective biodiversity conservation planning starts with genetic characterization within and among focal populations, in order to understand the likely impact of threats for ensuring the long-term viability of a species. The Wonder Gecko, Teratoscincus keyserlingii, is one of nine members of the genus. This species is distributed in Iran, Afghanistan, and Pakistan, with a small isolated population in the United Arab Emirates (UAE), where it is classified nationally as Critically Endangered. Within its Arabian range, anthropogenic activity is directly linked to the species' decline, with highly localised and severely fragmented populations. Here we describe the evolutionary history of Teratoscincus, by reconstructing its phylogenetic relationships and estimating its divergence times and ancestral biogeography. For conservation implications of T. keyserlingii we evaluate the genetic structure of the Arabian population using genomic data. This study supports the monophyly of most species and reveals considerable intraspecific variability in T. microlepis and T. keyserlingii, which necessitate broad systematic revisions. The UAE population of T. keyserlingii likely arrived from southern Iran during the Pleistocene and no internal structure was recovered within, implying a single population status. Regional conservation of T. keyserlingii requires improved land management and natural habitat restoration in the species' present distribution, and expansion of current protected areas, or establishment of new areas with suitable habitat for the species, mostly in northern Abu Dhabi Emirate.

Evidence of two deeply divergent co-existing mitochondrial genomes in the Tuatara reveals an extremely complex genomic organization.

Animal mitochondrial genomic polymorphism occurs as low-level mitochondrial heteroplasmy and deeply divergent co-existing molecules. The latter is rare, known only in bivalvian mollusks. Here we show two deeply divergent co-existing mt-genomes in a vertebrate through genomic sequencing of the Tuatara (Sphenodon punctatus), the sole-representative of an ancient reptilian Order. The two molecules, revealed using a combination of short-read and long-read sequencing technologies, differ by 10.4% nucleotide divergence. A single long-read covers an entire mt-molecule for both strands. Phylogenetic analyses suggest a 7-8 million-year divergence between genomes. Contrary to earlier reports, all 37 genes typical of animal mitochondria, with drastic gene rearrangements, are confirmed for both mt-genomes. Also unique to vertebrates, concerted evolution drives three near-identical putative Control Region non-coding blocks. Evidence of positive selection at sites linked to metabolically important transmembrane regions of encoded proteins suggests these two mt-genomes may confer an adaptive advantage for an unusually cold-tolerant reptile.

The tuatara genome reveals ancient features of amniote evolution.

The tuatara (Sphenodon punctatus)-the only living member of the reptilian order Rhynchocephalia (Sphenodontia), once widespread across Gondwana1,2-is an iconic species that is endemic to New Zealand2,3. A key link to the now-extinct stem reptiles (from which dinosaurs, modern reptiles, birds and mammals evolved), the tuatara provides key insights into the ancestral amniotes2,4. Here we analyse the genome of the tuatara, which-at approximately 5 Gb-is among the largest of the vertebrate genomes yet assembled. Our analyses of this genome, along with comparisons with other vertebrate genomes, reinforce the uniqueness of the tuatara. Phylogenetic analyses indicate that the tuatara lineage diverged from that of snakes and lizards around 250 million years ago. This lineage also shows moderate rates of molecular evolution, with instances of punctuated evolution. Our genome sequence analysis identifies expansions of proteins, non-protein-coding RNA families and repeat elements, the latter of which show an amalgam of reptilian and mammalian features. The sequencing of the tuatara genome provides a valuable resource for deep comparative analyses of tetrapods, as well as for tuatara biology and conservation. Our study also provides important insights into both the technical challenges and the cultural obligations that are associated with genome sequencing.

Publisher Correction: The tuatara genome reveals ancient features of amniote evolution.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Development of the Scalp Hair Assessment PRO™ measure for alopecia areata.

BACKGROUND: Valid patient-reported outcome (PRO) measures are required to evaluate alopecia areata (AA) treatments. OBJECTIVES: To develop a content-valid and clinically meaningful PRO measure to assess AA scalp hair loss with scores comparable with the five-response-level Alopecia Areata Investigator Global Assessment (AA-IGA™). METHODS: A draft PRO measure was developed based on input from 10 clinical experts in AA. The PRO measure was cognitively debriefed, modified and finalized through two rounds of qualitative semistructured interviews with patients with AA who had experienced ≥ 50% scalp hair loss. Data were thematically analysed. RESULTS: Adults (round 1: n = 25; round 2: n = 15) and adolescents aged 15-17 years (round 1: n = 5) in North America participated. All patients named scalp hair loss as a key AA sign or symptom. Patients demonstrated the ability to self-report their current amount of scalp hair using percentages. In round 1 not all patients interpreted the measurement concept consistently; therefore, the PRO was modified to clarify the measurement concept to improve usability. Following modifications, patients in round 2 responded without difficulty to the PRO measure. Patients confirmed that they could use the five-level response scale to rate their scalp hair loss: no missing hair, 0%; limited, 1-20%; moderate, 21-49%; large, 50-94%; nearly all or all, 95-100%. Almost all patients deemed hair regrowth resulting in ≤ 20% scalp hair loss a treatment success. CONCLUSIONS: The Scalp Hair Assessment PRO™ is a content-valid, clinically meaningful assessment of distinct gradations of scalp hair loss for evaluating AA treatment for patients with ≥ 50% hair loss at baseline.

The Alopecia Areata Investigator Global Assessment scale: a measure for evaluating clinically meaningful success in clinical trials.

BACKGROUND: Content-valid and clinically meaningful instruments are required to evaluate outcomes of therapeutic interventions in alopecia areata (AA). OBJECTIVES: To develop an Investigator's Global Assessment (IGA) to interpret treatment response in AA treatment studies. METHODS: Qualitative interviews were conducted in the USA with expert dermatologists and with patients with AA who had experienced ≥ 50% scalp-hair loss. Thematic data analysis identified critical outcomes and evaluated the content validity of the new IGA. RESULTS: Expert clinicians (n = 10) judged AA treatment success by the amount of scalp-hair growth (median 80% scalp hair). Adult (n = 25) and adolescent (n = 5) patients participated. Scalp-hair loss was the most bothersome AA sign/symptom for most patients. Perceived treatment success - short of 100% scalp hair - was the presence of ~ 70-90% scalp hair (median 80%). Using additional clinician and patient insights, the Alopecia Areata Investigator Global Assessment (AA-IGA™) was developed. This clinician-reported outcome assessment is an ordinal, static measure comprising five severity categories of scalp-hair loss. Nearly all clinicians and patients in this study agreed that, for patients with ≥ 50% scalp-hair loss, successful treatment would be hair regrowth resulting in ≤ 20% scalp-hair loss. CONCLUSIONS: We recommend using the Severity of Alopecia Tool to assess the extent (0-100%) of scalp-hair loss. The AA-IGA is a robust ordinal measure providing distinct and clinically meaningful gradations of scalp-hair loss that reflects patients' and expert clinicians' perspectives and treatment expectations. What is already known about this topic? The Severity of Alopecia Tool is widely used to assess the extent of scalp-hair loss in patients with alopecia areata. Guidelines define treatment success as a 50% improvement in scalp hair, and clinical trials have used dynamic thresholds of 50% and 90%. However, there is no clinical consensus on these endpoints, and patient perspectives on treatment success are unknown. What does this study add? Through qualitative interviews with 10 expert dermatologists and 30 patients with alopecia areata who had experienced ≥ 50% scalp-hair loss, we developed the Alopecia Areata Investigator Global Assessment (AA-IGA™) to measure five clinically meaningful gradations of alopecia areata scalp-hair loss that reflects patients' and clinicians' perspectives and expectations of treatment success in alopecia areata treatment studies. What are the clinical implications of this work? The AA-IGA is a robust ordinal measure that can inform clinical evaluation of alopecia areata treatment outcomes. The AA-IGA can be used to determine clinically meaningful treatment success for alopecia areata, with success defined by patients and clinicians as reaching ≤ 20% scalp-hair loss. Linked Comment: Blome. Br J Dermatol 2020; 183:609.

A molecular phylogenetic hypothesis for the Asian agamid lizard genus Phrynocephalus reveals discrete biogeographic clades implicated by plate tectonics.

Phylogenetic relationships of the agamid lizard genus Phrynocephalus are described in the context of plate tectonics. A near comprehensive taxon sampling reports three data sets: (1) mitochondrial DNA from ND1 to COI (3' end of ND1, tRNAGln, tRNAIle, tRNAMet, ND2, tRNATrp, tRNAAla, tRNAAsn, tRNACys, tRNATyr, and the 5' end of COI) with 1761 aligned positional sites (1595 included, 839 informative), (2) nuclear RAG-1 DNA with 2760 aligned positional sites (342 informative), and (3) 25 informative allozyme loci with 213 alleles (107 informative when coded as presence/absence). It is hypothesized that Phrynocephalus phyletic patterns and speciation reflect fault lines of ancient plates now in Asia rejuvenated by the more recent Indian and Arabian plate collisions. Molecular estimates of lineage splits are highly congruent with geologic dates from the literature.  A southern origin for the genus in Southwest Asia is resolved in phylogenetic estimates and a northern origin is statistically rejected. On the basis of monophyly and molecular evidence several taxa previously recognized as subspecies are recognized as species: P. hongyuanensis, P. sogdianus, and P. strauchi as "Current Status"; Phrynocephalus bannikovi, Phrynocephalus longicaudatus, Phrynocephalus turcomanus, and Phrynocephalus vindumi are formally "New Status". Phylogenetic evaluation indicates a soft substrate habitat of sand for the shared ancestor of modern Phrynocephalus. Size diversity maximally overlaps in the Caspian Basin and northwestern Iranian Plateau. The greatest species numbers of six in sympatry and regional allopatry are found in the southern Caspian Basin and southern Helmand Basin, both from numerous phylogenetic lineages in close proximity attributed to tectonic induced events.

[MRI of the pulmonary parenchyma: Towards clinical applicability?] / IRM du parenchyme pulmonaire : vers une application clinique ?

Lung parenchyma has long been considered out of the scope of magnetic resonance imaging (MRI) clinical applicability. However, technological advances have emerged to soluce the technical difficulties and thus, applications in clinical practice have become realistic. Nevertheless, various approaches have been proposed and there is a need to synthetize the most recent literature data in order to envision a rationale to build lung MR protocols for clinical use. In addition, these technological innovations may modify the usual paradigms of lung MRI, which are still not consensual. Thus, lung MR protocols appear to be heterogeneous across expert centers in the current context. In this literature review, we ought to describe a rationale on the need to get an alternative to ionizing imaging modalities, in particular in the follow-up of patients with chronic lung diseases. We will describe the most recent technical advances regarding both morphological and functional MRI. Finally, we will conclude on the clinical applicability of MRI of the pulmonary parenchyma, as a routine or research tool.

[New therapeutic developments in cystic fibrosis]. / Nouvelles thérapeutiques ciblant le canal chlorure dans la mucoviscidose.

Since the discovery of chloride secretion by the Cystic Fibrosis Transport regulator CFTR in 1983, and CFTR gene in 1989, knowledge about CFTR synthesis, maturation, intracellular transfer and function has dramatically expanded. These discoveries have led to the distribution of CF mutations into 6 classes with different pathophysiological mechanisms. In this article we will explore the state of art on CFTR synthesis and its chloride secretion function. We will then explore the consequences of the 6 classes of mutations on CFTR protein function and we will describe the new therapeutic developments aiming at correcting these defects.

Exploring the expression of Pseudomonas aeruginosa genes directly from sputa of cystic fibrosis patients.

UNLABELLED: Acquisition of Pseudomonas aeruginosa is known as a negative prognostic factor in patients with cystic fibrosis. We started a pilot study to evaluate Ps. aeruginosa gene expression directly from the sputum of infected patients. Total RNA was purified from 15 sputum samples collected from 10 patients, and the expression levels of five genes from Ps. aeruginosa were measured by RT-qPCR. Expression of algD, algR, antB, lasB and pqsA genes was determined in sputa that contained Ps. aeruginosa cells. The resultant data provided an overview of the expression of these genes in CF patients. Except for the correlation between algD expression and the mucoid phenotype, the gene expression profile could not be associated with the clinical status of patients. However, beyond the heterogeneity of the Ps. aeruginosa phenotype in sputum, we observed a correlation between the expression of antB and pqsA and a low level of lasB transcripts. SIGNIFICANCE AND IMPACT OF THE STUDY: Pseudomonas aeruginosa infection leads to high morbidity and mortality in cystic fibrosis patients. The identification of Ps. aeruginosa-assigned factors is important to eradicate the colonization. We started a pilot study to evaluate the gene expression of Ps. aeruginosa directly from the sputum of infected patients. Preliminary results suggest that beyond the heterogeneity of the Ps. aeruginosa phenotype in sputum, we observe a correlation between the expression of antB and pqsA and a low level of lasB transcripts. This approach could shed some light on the behaviour of Ps. aeruginosa during pulmonary infection and may reveal some important elements for optimizing therapy.

Molecular phylogenetic reconstruction of the endemic Asian salamander family Hynobiidae (Amphibia, Caudata).

The salamander family Hynobiidae contains over 50 species and has been the subject of a number of molecular phylogenetic investigations aimed at reconstructing branches across the entire family. In general, studies using the greatest amount of sequence data have used reduced taxon sampling, while the study with the greatest taxon sampling has used a limited sequence data set. Here, we provide insights into the phylogenetic history of the Hynobiidae using both dense taxon sampling and a large mitochondrial DNA sequence data set. We report exclusive new mitochondrial DNA data of 2566 aligned bases (with 151 excluded sites, of included sites 1157 are variable with 957 parsimony informative). This is sampled from two genic regions encoding a 12S-16S region (the 3' end of 12S rRNA, tRNA(VAI), and the 5' end of 16S rRNA), and a ND2-COI region (ND2, tRNA(Trp), tRNA(Ala), tRNA(Asn), the origin for light strand replication--O(L), tRNA(Cys), tRNAT(Tyr), and the 5' end of COI). Analyses using parsimony, Bayesian, and maximum likelihood optimality criteria produce similar phylogenetic trees, with discordant branches generally receiving low levels of branch support. Monophyly of the Hynobiidae is strongly supported across all analyses, as is the sister relationship and deep divergence between the genus Onychodactylus with all remaining hynobiids. Within this latter grouping our phylogenetic results identify six clades that are relatively divergent from one another, but for which there is minimal support for their phylogenetic placement. This includes the genus Batrachuperus, the genus Hynobius, the genus Pachyhynobius, the genus Salamandrella, a clade containing the genera Ranodon and Paradactylodon, and a clade containing the genera Liua and Pseudohynobius. This latter clade receives low bootstrap support in the parsimony analysis, but is consistent across all three analytical methods. Our results also clarify a number of well-supported relationships within the larger Batrachuperus and Hynobius clades. While the relationships identified in this study do much to clarify the phylogenetic history of the Hynobiidae, the poor resolution among major hynobiid clades, and the contrast of mtDNA-derived relationships with recent phylogenetic results from a small number of nuclear genes, highlights the need for continued phylogenetic study with larger numbers of nuclear loci.

[Surgical lung biopsy: Indications and therapeutic implications]. / Biopsie pulmonaire chirurgicale : indications et incidences thérapeutiques.

Surgical biopsy of lung parenchyma can be used to establish a diagnosis in interstitial lung disease both of acute and chronic presentation. The present article summarizes the current indications, the therapeutic implications, the different surgical techniques and postoperative complications of the procedure. Common controversies and problems related to surgical lung biopsy are also presented.

Pseudomonas aeruginosa induces vascular endothelial growth factor synthesis in airway epithelium in vitro and in vivo.

Pseudomonas aeruginosa (PA) airway infection and bronchial blood vessel proliferation are features of bronchiectasis. Because vascular endothelial growth factor (VEGF)-A regulates angiogenesis, we hypothesised that PA infection induces VEGF synthesis in epithelium and peribronchial angiogenesis. Because epidermal growth factor receptor (EGFR) activation regulates VEGF synthesis in cancer, we also evaluated the roles of EGFR. Airway epithelial cells were incubated for 24 h with PA supernatants and VEGF concentrations were measured in culture medium by ELISA. C57BL/6N mice were instilled intratracheally with sterile agarose beads or with agarose beads coated with the PA strain PAO1 (mean ± sem 6 × 10(5) ± 3 × 10(5) cfu · animal(-1)), with or without the EGFR inhibitor AG1478 (12.5 mg · kg(-1) · day(-1) intraperitoneally). Epithelial immunostaining for VEGF and phosphorylated EGFR, and peribronchial vascularity, were quantified using morphometric analysis. VEGF expression was further assessed by western blot in mouse lung homogenates. PA supernatants induced dose-dependent VEGF synthesis in cultured airway epithelial cells, effects which were prevented by EGFR antagonists. In mice, persistent PAO1 infection increased immunostaining for VEGF and phosphorylated EGFR in airway epithelium, and resulted in increased peribronchial vascularity within 7 days. These effects were reduced by EGFR inhibition. Persistent PA infection induced VEGF synthesis in airway epithelium and peribronchial angiogenesis, at least in part via EGFR-dependent mechanisms.

Vicariance and dispersal form a ring distribution in nightsnakes around the Gulf of California.

Plate tectonics can have profound effects on organismal distribution and is often the driving force in speciation. Through geologic processes, the Baja California Peninsula depicts two faunal patterns: one through southern vicariance with Cape separation, and the other through dispersal onto the northern peninsula, referred to as a 'dual-peninsular effect.' Here we apply a hierarchical sampling strategy that combines population-level sequence data ( approximately 800bp, nad4 region) with complete mt-genome data (aligned 15,549bp) and 5 nuclear protein encoding loci (3315bp), to test whether both patterns have occurred in one group of nightsnakes (Hypsiglena). The geologic formation of the peninsula is thought to have occurred in three stages: (1) Cape separation from mainland Mexico; (2) the northern peninsula separated, forming the northern Gulf of California; and (3) the peninsula was united through volcanic activity, while moving northward causing collision with southern California. However, the timing of events is debated. We explore phylogenetic relationships and estimate dates of divergence for nightsnakes using our hierarchical sampling strategy. Our data support both 'southern-vicariance' and 'northern-dispersal' have occurred in nightsnakes, forming a ring distribution around the Gulf of California. Two divergent forms are sympatric on the southern half of the peninsula with no indication of hybridization. Nightsnakes represent the first group to depict the 'dual-peninsular effect' with extensive overlap on the Baja California Peninsula.