­­Atg9 interactions via its transmembrane domains are required for phagophore expansion during autophagy

During macroautophagy/autophagy, precursor cisterna known as phagophores expand and sequester portions of the cytoplasm and/or organelles, and subsequently close resulting in double-membrane transport vesicles called autophagosomes. Autophagosomes fuse with lysosomes/vacuoles to allow the degradation and recycling of their cargoes. We previously showed that sequential binding of yeast Atg2 and Atg18 to Atg9, the only conserved transmembrane protein in autophagy, at the extremities of the phagophore mediates the establishment of membrane contact sites between the phagophore and the endoplasmic reticulum. As the Atg2-Atg18 complex transfers lipids between adjacent membranes in vitro, it has been postulated that this activity and the scramblase activity of the trimers formed by Atg9 are required for the phagophore expansion. Here, we present evidence that Atg9 indeed promotes Atg2-Atg18 complex-mediated lipid transfer in vitro, although this is not the only requirement for its function in vivo. In particular, we show that Atg9 function is dramatically compromised by a F627A mutation within the conserved interface between the transmembrane domains of the Atg9 monomers. Although Atg9F627A self-interacts and binds to the Atg2-Atg18 complex, the F627A mutation blocks the phagophore expansion and thus autophagy progression. This phenotype is conserved because the corresponding human ATG9A mutant severely impairs autophagy as well. Importantly, Atg9F627A has identical scramblase activity in vitro like Atg9, and as with the wild-type protein enhances Atg2-Atg18-mediated lipid transfer. Collectively, our data reveal that interactions of Atg9 trimers via their transmembrane segments play a key role in phagophore expansion beyond Atg9ʹs role as a lipid scramblase.Abbreviations: BafA1: bafilomycin A1; Cvt: cytoplasm-to-vacuole targeting; Cryo-EM: cryo-electron microscopy; ER: endoplasmic reticulum; GFP: green fluorescent protein; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MCS: membrane contact site; NBD-PE: N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine; PAS: phagophore assembly site; PE: phosphatidylethanolamine; prApe1: precursor Ape1; PtdIns3P: phosphatidylinositol-3-phosphate; SLB: supported lipid bilayer; SUV: small unilamellar vesicle; TMD: transmembrane domain; WT: wild type.</p

BFL1 Modulates Apoptosis at the Membrane Level through a Bifunctional And Multimodal Mechanism Showing Key Differences With BCLXL

BFL1 is a relatively understudied member of the BCL2 protein family which has been implicated in the pathogenesis andchemoresistance of a variety of human cancers, including hematological malignancies and solid tumours. BFL1 is generallyconsidered to have an antiapoptotic function, although its precise mode of action remains unclear. By quantitativelyanalyzing BFL1 action in synthetic membrane models and in cells, we found that BFL1 inhibits apoptosis through threedistinct mechanisms which are similar but not identical to those of BCLXL, the paradigmatic antiapoptotic BCL2 familyprotein. Strikingly, alterations in lipid composition during apoptosis activate a prodeath function of BFL1 that is based onnoncanonical oligomerization of the protein and breaching of the permeability barrier of the outer mitochondrial membrane(OMM). This lipid-triggered prodeath function of BFL1 is absent in BCLXL and also differs from that of the apoptoticeffector BAX, which sets it apart from other BCL2 family members. Ourfindings support a new model in which BFL1modulates apoptosis through a bifunctional and multimodal mode of action that is distinctly regulated by OMM lipidscompared to BCLXL.This work was supported by Grants BFU2011-28566 from the Ministerio de Economia y Competitividad and IT838-13 from Gobierno Vasco. HFR is a recipient of a predoctoral fellowship from the Ministerio de Educacion (Spain). We also thank to LE facility technician in the Achucarro Basque Center for Neuroscience for the support in STED experiments. Finally, we thank Dr. Frank Essmann and Prof. Klaus Schulze-Osthoff for providing the HCT116 BAX/BAK DKO cells and Prof. Jean Claude Martinou for HCT116 CL KO cells

ZNF521 Enhances MLL-AF9-Dependent Hematopoietic Stem Cell Transformation in Acute Myeloid Leukemias by Altering the Gene Expression Landscape

Leukemias derived from the MLL-AF9 rearrangement rely on dysfunctional transcriptional networks. ZNF521, a transcription co-factor implicated in the control of hematopoiesis, has been proposed to sustain leukemic transformation in collaboration with other oncogenes. Here, we demonstrate that ZNF521 mRNA levels correlate with specific genetic aberrations: in particular, the highest expression is observed in AMLs bearing MLL rearrangements, while the lowest is detected in AMLs with FLT3-ITD, NPM1, or CEBPα double mutations. In cord blood-derived CD34(+) cells, enforced expression of ZNF521 provides a significant proliferative advantage and enhances MLL-AF9 effects on the induction of proliferation and the expansion of leukemic progenitor cells. Transcriptome analysis of primary CD34(+) cultures displayed subsets of genes up-regulated by MLL-AF9 or ZNF521 single transgene overexpression as well as in MLL-AF9/ZNF521 combinations, at either the early or late time points of an in vitro leukemogenesis model. The silencing of ZNF521 in the MLL-AF9 + THP-1 cell line coherently results in an impairment of growth and clonogenicity, recapitulating the effects observed in primary cells. Taken together, these results underscore a role for ZNF521 in sustaining the self-renewal of the immature AML compartment, most likely through the perturbation of the gene expression landscape, which ultimately favors the expansion of MLL-AF9-transformed leukemic clones

Systematic Assessment of the Accuracy of Subunit Counting in Biomolecular Complexes Using Automated Single-Molecule Brightness Analysis.

Analysis of single-molecule brightness allows subunit counting of high-order oligomeric biomolecular complexes. Although the theory behind the method has been extensively assessed, systematic analysis of the experimental conditions required to accurately quantify the stoichiometry of biological complexes remains challenging. In this work, we develop a high-throughput, automated computational pipeline for single-molecule brightness analysis that requires minimal human input. We use this strategy to systematically quantify the accuracy of counting under a wide range of experimental conditions in simulated ground-truth data and then validate its use on experimentally obtained data. Our approach defines a set of conditions under which subunit counting by brightness analysis is designed to work optimally and helps in establishing the experimental limits in quantifying the number of subunits in a complex of interest. Finally, we combine these features into a powerful, yet simple, software that can be easily used for the analysis of the stoichiometry of such complexes

Depressão em idosos de uma instituição de longa permanência

Objective: to determine the prevalence of depression in elderly residents of a Long Stay Institution for Aged. Method: this is a descriptive study transverse, of quantitative approach. The population consisted of 48 subjects. To assess the mental state was used Mini Mental State Examination and collection of data to the Geriatric Depression Scale (GDS-15). Were ethical principles contained in Resolution 196/96 of the National Health. Results: results showed that 25% of the elderly showed no signs of depression, 43.75% showed signs of depression, mild/moderate and 31.25% showed signs of severe depression. The prevalence of depression was higher in women (55.5%) than men (44.5%). Conclusion: high levels of depression in the sample report identified the need for psychological support planning can provide a better quality of life for residents in institution studied as well as warn of similar prevalence in the general ILPIsObjetivo: determinar la prevalencia de depresión en los ancianos residentes de una institución a largo plazo para los Ancianos (ILPI). Metodología: se realizó un estudio descriptivo, transverso, de enfoque cuantitativo. La población estuvo conformada por 48 sujetos. Para evaluar el estado mental se utilizó Mini Mental State Examination y la depresión con la Escala de Depresión Geriátrica (GDS-15). Resultados: los resultados mostraron que el 25% de los ancianos no mostró signos de depresión, 43,75% mostraron señales de depresión, leve/moderada y 31,25% mostraron signos de la depresión severa. La prevalencia de depresión fue mayor en mujeres (55,5%) que hombres (44,5%). Conclusión: los niveles altos de depresión en el informe de la muestra identificó la necesidad de planificar el apoyo psicológico puede proporcionar una mejor calidad de vida de los residentes en la institución estudiada, así como advertir de prevalencia similares en los ILPI generales.Objetivo: determinar a prevalência de depressão em idosos residentes em uma Instituição de Longa Permanência para Idosos (ILPI). Metodologia: trata-se de um estudo descritivo de abordagem quantitativa. A amostra foi constituída de 48 sujeitos. Para a avaliação do estado mental foi aplicado o Mini Mental State Examination e para a depressão a Escala de Depressão Geriátrica (EDG-15). Foram seguidos os princípios éticos da Resolução 196/96 do Conselho Nacional da Saúde. Resultados: os resultados demonstraram que 25% dos idosos não apresentaram sinais de depressão; 43,75% apresentaram sinais de depressão leve/moderada e 31,25% apresentaram sinais de depressão severa. A prevalência de depressão foi maior em mulheres (55,5%) do que em homens (44,5%). Conclusão: os altos níveis de depressão identificados na amostra reportam para a necessidade de planejamento de apoio psicológico capaz de proporcionar melhor qualidade de vida aos residentes na ILPI estudada, como também alertam para semelhante prevalência nas ILPIs em geral

Five carbon- and nitrogen-bearing species in a hot giant planet's atmosphere

The atmospheres of gaseous giant exoplanets orbiting close to their parent stars (hot Jupiters) have been probed for nearly two decades. They allow us to investigate the chemical and physical properties of planetary atmospheres under extreme irradiation conditions. Previous observations of hot Jupiters as they transit in front of their host stars have revealed the frequent presence of water vapour and carbon monoxide in their atmospheres; this has been studied in terms of scaled solar composition under the usual assumption of chemical equilibrium. Both molecules as well as hydrogen cyanide were found in the atmosphere of HD 209458b, a well studied hot Jupiter (with equilibrium temperature around 1,500 kelvin), whereas ammonia was tentatively detected there and subsequently refuted. Here we report observations of HD 209458b that indicate the presence of water (H2O), carbon monoxide (CO), hydrogen cyanide (HCN), methane (CH4), ammonia (NH3) and acetylene (C2H2), with statistical significance of 5.3 to 9.9 standard deviations per molecule. Atmospheric models in radiative and chemical equilibrium that account for the detected species indicate a carbon-rich chemistry with a carbon-to-oxygen ratio close to or greater than 1, higher than the solar value (0.55). According to existing models relating the atmospheric chemistry to planet formation and migration scenarios, this would suggest that HD 209458b formed far from its present location and subsequently migrated inwards. Other hot Jupiters may also show a richer chemistry than has been previously found, which would bring into question the frequently made assumption that they have solar-like and oxygen-rich compositions.Comment: As part of the Springer Nature Content Sharing Initiative, it is possible to access a view-only version of this paper by using the following SharedIt link: https://rdcu.be/cifr

HADES RV Programme with HARPS-N at TNG . III. Flux-flux and activity-rotation relationships of early-M dwarfs

Context. Understanding stellar activity in M dwarfs is crucial for the physics of stellar atmospheres and for ongoing radial velocity exoplanet programmes. Despite the increasing interest in M dwarfs, our knowledge of the chromospheres of these stars is far from being complete. Aims: We test whether the relations between activity, rotation, and stellar parameters and flux-flux relationships previously investigated for main-sequence FGK stars and for pre-main-sequence M stars also hold for early-M dwarfs on the main-sequence. Although several attempts have been made so far, here we analyse a large sample of stars undergoing relatively low activity. Methods: We analyse in a homogeneous and coherent way a well-defined sample of 71 late-K/early-M dwarfs that are currently being observed in the framework of the HArps-N red Dwarf Exoplanet Survey (HADES). Rotational velocities are derived using the cross-correlation technique, while emission flux excesses in the Ca II H & K and Balmer lines from Hα up to H∊ are obtained by using the spectral subtraction technique. The relationships between the emission excesses and the stellar parameters (projected rotational velocity, effective temperature, kinematics, and age) are studied. Relations between pairs of fluxes of different chromospheric lines (flux-flux relationships) are also studied and compared with the literature results for other samples of stars. Results: We find that the strength of the chromospheric emission in the Ca II H & K and Balmer lines is roughly constant for stars in the M0-M3 spectral range. Although our sample is likely to be biased towards inactive stars, our data suggest that a moderate but significant correlation between activity and rotation might be present, as well as a hint of kinematically selected young stars showing higher levels of emission in the calcium line and in most of the Balmer lines. We find our sample of M dwarfs to be complementary in terms of chromospheric and X-ray fluxes with those of the literature, extending the analysis of the flux-flux relationships to the very low flux domain. Conclusions: Our results agree with previous works suggesting that the activity-rotation-age relationship known to hold for solar-type stars also applies to early-M dwarfs. We also confirm previous findings that the field stars which deviate from the bulk of the empirical flux-flux relationships show evidence of youth. Based on observations made with the Italian Telescopio Nazionale Galileo (TNG), operated on the island of La Palma by the Fundación Galileo Galilei of the INAF (Istituto Nazionale di Astrofisica) at the Spanish Observatorio del Roque de los Muchachos of the Instituto de Astrofísica de Canarias

The GAPS programme with HARPS-N at TNG. X. Differential abundances in the XO-2 planet-hosting binary

Binary stars hosting exoplanets are a unique laboratory where chemical tagging can be performed to measure the elemental abundances of both stellar components with high accuracy, with the aim to investigate the formation of planets and their subsequent evolution. Here, we present a high-precision differential abundance analysis of the XO-2 wide stellar binary based on high-resolution HARPS-N at TNG spectra. Both components are very similar K-dwarfs and host planets. Since they formed presumably within the same molecular cloud, we expect that they possess the same initial elemental abundances. We investigated whether planets can cause some chemical imprints in the stellar atmospheric abundances. We measure abundances of 25 elements for both stars with a range of condensation temperature TC = 40-1741 K, achieving typical precisions of ~0.07 dex. The northern component shows abundances in all elements higher by +0.067 ± 0.032 dex on average, with a mean difference of +0.078 dex for elements with TC > 800 K. The significance of the XO-2N abundance difference relative to XO-2S is at the 2σ level for almost all elements. We discuss that this result might be interpreted as the signature of the ingestion of material by XO-2N or depletion in XO-2S that is due to locking of heavy elements by the planetary companions. We estimate a mass of several tens of M⊕ in heavy elements. The difference in abundances between XO-2N and XO-2S shows a positive correlation with the condensation temperatures of the elements, with a slope of (4.7 ± 0.9) × 10-5 dex K-1, which could mean that both components have not formed terrestrial planets, but first experienced the accretion of rocky core interior to the subsequent giant planets. Based on observations made with the Italian Telescopio Nazionale Galileo (TNG), operated on the island of La Palma by the INAF - Fundación Galileo Galilei at the Roche de los Muchachos Observatory of the Instituto de Astrofísica de Canarias (IAC) in the framework of the large programme Global Architecture of Planetary Systems (GAPS; P.I. A. Sozzetti).Final reduced spectra (FITS) are only available at the CDS via anonymous ftp to http://cdsarc.u-strasbg.fr (ftp://130.79.128.5) or via http://cdsarc.u-strasbg.fr/viz-bin/qcat?J/A+A/583/A135</A

The GAPS Programme at TNG XXXIX. Multiple molecular species in the atmosphere of the warm Giant Planet WASP-80 b unveiled at high resolution with GIANO-B*

Detections of molecules in the atmosphere of gas giant exoplanets allow us to investigate the physico-chemical properties of the atmospheres. Their inferred chemical composition is used as tracer of planet formation and evolution mechanisms. Currently, an increasing number of detections is showing a possible rich chemistry of the hotter gaseous planets, but whether this extends to cooler giants is still unknown. We observed four transits of WASP-80 b, a warm transiting giant planet orbiting a late-K dwarf star with the near-infrared GIANO-B spectrograph installed at the Telescopio Nazionale Galileo and performed high-resolution transmission spectroscopy analysis. We report the detection of several molecular species in its atmosphere. Combining the four nights and comparing our transmission spectrum to planetary atmosphere models containing the signature of individual molecules within the cross-correlation framework, we find the presence of H2O, CH4, NH3, and HCN with high significance, tentative detection of CO2, and inconclusive results for C2H2 and CO. A qualitative interpretation of these results, using physically motivated models, suggests an atmosphere consistent with solar composition and the presence of disequilibrium chemistry and we therefore recommend the inclusion of the latter in future modeling of sub-1000 K planets