Searching for novel gene functions in yeast : identification of thousands of novel molecular interactions by protein-fragment complementation assay followed by automated gene function prediction and high-throughput lipidomics

La compréhension de processus biologiques complexes requiert des approches expérimentales et informatiques sophistiquées. Les récents progrès dans le domaine des stratégies génomiques fonctionnelles mettent dorénavant à notre disposition de puissants outils de collecte de données sur l’interconnectivité des gènes, des protéines et des petites molécules, dans le but d’étudier les principes organisationnels de leurs réseaux cellulaires. L’intégration de ces connaissances au sein d’un cadre de référence en biologie systémique permettrait la prédiction de nouvelles fonctions de gènes qui demeurent non caractérisées à ce jour. Afin de réaliser de telles prédictions à l’échelle génomique chez la levure Saccharomyces cerevisiae, nous avons développé une stratégie innovatrice qui combine le criblage interactomique à haut débit des interactions protéines-protéines, la prédiction de la fonction des gènes in silico ainsi que la validation de ces prédictions avec la lipidomique à haut débit. D’abord, nous avons exécuté un dépistage à grande échelle des interactions protéines-protéines à l’aide de la complémentation de fragments protéiques. Cette méthode a permis de déceler des interactions in vivo entre les protéines exprimées par leurs promoteurs naturels. De plus, aucun biais lié aux interactions des membranes n’a pu être mis en évidence avec cette méthode, comparativement aux autres techniques existantes qui décèlent les interactions protéines-protéines. Conséquemment, nous avons découvert plusieurs nouvelles interactions et nous avons augmenté la couverture d’un interactome d’homéostasie lipidique dont la compréhension demeure encore incomplète à ce jour. Par la suite, nous avons appliqué un algorithme d’apprentissage afin d’identifier huit gènes non caractérisés ayant un rôle potentiel dans le métabolisme des lipides. Finalement, nous avons étudié si ces gènes et un groupe de régulateurs transcriptionnels distincts, non préalablement impliqués avec les lipides, avaient un rôle dans l’homéostasie des lipides. Dans ce but, nous avons analysé les lipidomes des délétions mutantes de gènes sélectionnés. Afin d’examiner une grande quantité de souches, nous avons développé une plateforme à haut débit pour le criblage lipidomique à contenu élevé des bibliothèques de levures mutantes. Cette plateforme consiste en la spectrométrie de masse à haute resolution Orbitrap et en un cadre de traitement des données dédié et supportant le phénotypage des lipides de centaines de mutations de Saccharomyces cerevisiae. Les méthodes expérimentales en lipidomiques ont confirmé les prédictions fonctionnelles en démontrant certaines différences au sein des phénotypes métaboliques lipidiques des délétions mutantes ayant une absence des gènes YBR141C et YJR015W, connus pour leur implication dans le métabolisme des lipides. Une altération du phénotype lipidique a également été observé pour une délétion mutante du facteur de transcription KAR4 qui n’avait pas été auparavant lié au métabolisme lipidique. Tous ces résultats démontrent qu’un processus qui intègre l’acquisition de nouvelles interactions moléculaires, la prédiction informatique des fonctions des gènes et une plateforme lipidomique innovatrice à haut débit , constitue un ajout important aux méthodologies existantes en biologie systémique. Les développements en méthodologies génomiques fonctionnelles et en technologies lipidomiques fournissent donc de nouveaux moyens pour étudier les réseaux biologiques des eucaryotes supérieurs, incluant les mammifères. Par conséquent, le stratégie présenté ici détient un potentiel d’application au sein d’organismes plus complexes.Understanding complex biological processes requires sophisticated experimental and computational approaches. The advances in functional genomics strategies provide powerful tools for collecting diverse types of information on interconnectivity of genes, proteins and small molecules for studying organizational principles of cellular networks. Integration of that knowledge into a systems biology framework enables prediction of novel functions of uncharacterized genes. For performing such predictions on a genome-wide scale in the yeast Saccharomyces cerevisiae, we have developed a novel strategy that combines high-throughput interactomics screen for protein-protein interactions, in silico gene function prediction, and validation of predictions with high-throughput lipidomics. We started by performing a large-scale screen for protein-protein interactions using a protein-fragment complementation assay. The method allowed to monitor interactions in vivo between proteins expressed from their natural promoters. Furthermore, the method did not suffer from bias against membrane interactions comparing to established genome-wide techniques for detecting protein interactions. As a result, we detected many novel interactions and increased coverage of an interactome of lipid homeostasis that has not been yet comprehensively explored. Next, we applied a machine learning algorithm to identify eight previously uncharacterized genes with a potential role in lipid metabolism. Finally, we investigated whether these genes and a set of distinct transcriptional regulators, not implicated previously with lipids, have a role in lipid homeostasis. For that purpose, we analyzed lipidome of deletion mutants of the selected genes. In order to probe a large number of strains, we have developed a high-throughput platform for high-content lipidomic screening of yeast mutant libraries that consists of high-resolution Orbitrap mass spectrometry and a dedicated data processing framework to support lipid phenotyping across hundreds of Saccharomyces cerevisiae mutants. Lipidomics experiments confirmed functional predictions by demonstrating differences of the lipid metabolic phenotypes of deletion mutants lacking YBR141C and YJR015W genes predicted to be involved in lipid metabolism. An altered lipid phenotype was also observed for a deletion mutant of the transcription factor KAR4 that has not been linked previously with lipid metabolism. These results demonstrate that a workflow that integrates the acquisition of novel molecular interactions, computational gene function prediction and novel high-throughput shotgun lipidomics platform is a valuable contribution to an arsenal of methods for systems biology. The developments of functional genomic methods and lipidomics technologies provide means to study biological networks of higher eukaryotes, including mammals. Therefore, the presented workflow has a potential to find its applications in more complex organisms

Randomized clinical trial of surgical vs. percutaneous vs. hybrid revascularization in multivessel coronary artery disease : residual myocardial ischemia and clinical outcomes at one year : Hybrid coronary REvascularization Versus Stenting or Surgery (HREVS)

Aim. Optimal revascularization strategy in multivessel (MV) coronary artery disease (CAD) eligible for percutaneous management (PCI) and surgery remains unresolved. We evaluated, in a randomized clinical trial, residual myocardial ischemia (RI) and clinical outcomes of MV-CAD revascularization using coronary artery bypass grafting (CABG), hybrid coronary revascularization (HCR), or MV-PCI. Methods. Consecutive MV-CAD patients (n = 155) were randomized (1 : 1 : 1) to conventional CABG (LIMA-LAD plus venous grafts) or HCR (MIDCAB LIMA-LAD followed by PCI for remaining vessels) or MV-PCI (everolimus-eluting CoCr stents) under Heart Team agreement on equal technical and clinical feasibility of each strategy. SPECT at 12 months (primary endpoint of RI that the trial was powered for; a measure of revascularization midterm efficacy and an independent predictor of long-term prognosis) preceded routine angiographic control. Results. Data are given, respectively, for the CABG, HCR, and MV-PCI arms. Incomplete revascularization rate was 8.0% vs. 7.7% vs. 5.7% (p=0.71). Hospital stay was 13.8 vs. 13.5 vs. 4.5 days (p<0.001), and sick-leave duration was 23 vs. 16 vs. 8 weeks (p<0.001). At 12 months, RI was 5 (2, 9)% vs. 5 (3, 7)% vs. 6 (3, 10)% (median; Q1, Q3) with noninferiority p values of 0.0006 (HCR vs. CABG) and 0.016 (MV-PCI vs. CABG). Rates of angiographic graft stenosis/occlusion or in-segment restenosis were 20.4% vs. 8.2% vs. 5.9% (p=0.05). Clinical target vessel/graft failure occurred in 12.0% vs. 11.5% vs. 11.3% (p=0.62). Major adverse cardiac and cerebral event (MACCE) rate was similar (12% vs. 13.4% vs. 13.2%; p=0.83). Conclusion. In this first randomized controlled study comparing CABG, HCR, and MV-PCI, residual myocardial ischemia and MACCE were similar at 12 months. There was no midterm indication of any added value of HCR. Hospital stay and sick-leave duration were shortest with MV-PCI. While longer-term follow-up is warranted, these findings may impact patient and physician choices and healthcare resources utilization. This trial is registered with NCT01699048

Magnetic Dirac semimetal state of (Mn,Ge)Bi2_2Te4_4

For quantum electronics, the possibility to finely tune the properties of magnetic topological insulators (TIs) is a key issue. We studied solid solutions between two isostructural Z$_2$ TIs, magnetic MnBi$_2$Te$_4$ and nonmagnetic GeBi$_2$Te$_4$, with Z$_2$ invariants of 1;000 and 1;001, respectively. For high-quality, large mixed crystals of Ge$_x$Mn$_{1-x}$Bi$_2$Te$_4$, we observed linear x-dependent magnetic properties, composition-independent pairwise exchange interactions along with an easy magnetization axis. The bulk band gap gradually decreases to zero for $x$ from 0 to 0.4, before reopening for $x>0.6$, evidencing topological phase transitions (TPTs) between topologically nontrivial phases and the semimetal state. The TPTs are driven purely by the variation of orbital contributions. By tracing the x-dependent $6p$ contribution to the states near the fundamental gap, the effective spin-orbit coupling variation is extracted. As $x$ varies, the maximum of this contribution switches from the valence to the conduction band, thereby driving two TPTs. The gapless state observed at $x=0.42$ closely resembles a Dirac semimetal above the Neel temperature and shows a magnetic gap below, which is clearly visible in raw photoemission data. The observed behavior of the Ge$_x$Mn$_{1-x}$Bi$_2$Te$_4$ system thereby demonstrates an ability to precisely control topological and magnetic properties of TIs

Site- and spin-dependent coupling at the highly ordered h-BN/Co(0001) interface

Using photoelectron diffraction and spectroscopy, we explore the structural and electronic properties of the hexagonal boron nitride (h-BN) monolayer epitaxially grown on the Co(0001) surface. Perfect matching of the lattice parameters allows formation of a well-defined interface where the B atoms occupy the hollow sites while the N atoms are located above the Co atoms. The corrugation of the h-BN monolayer and its distance from the substrate were determined by means of R-factor analysis. The obtained results are in perfect agreement with the density functional theory (DFT) predictions. The electronic structure of the interface is characterized by a significant mixing of the h-BN and Co states. Such hybridized states appear in the h-BN band gap. This allows to obtain atomically resolved scanning tunneling microscopy (STM) images from the formally insulating 2D material being in contact with ferromagnetic metal. The STM images reveal mainly the nitrogen sublattice due to a dominating contribution of nitrogen orbitals to the electronic states at the Fermi level. We believe that the high quality, well-defined structure and interesting electronic properties make the h-BN/Co(0001) interface suitable for spintronic applications.L.V.Ya. acknowledges the RSF (Grant No. 16-42-01093). A.V.T., V.O.S., K.A.B., O.Yu.V., and D.Yu.U. acknowledge St. Petersburg State University for research Grant No. 11.65.42.2017. M.V.K. and I.I.O. acknowledge the RFBR (Grant No. 16-29-06410). C.L. acknowledges the DFG (Grant Nos. LA655-17/1 and LA655-19/1).Peer reviewe

Lipidomics: A Tool for Studies of Atherosclerosis

Lipids, abundant constituents of both the vascular plaque and lipoproteins, play a pivotal role in atherosclerosis. Mass spectrometry-based analysis of lipids, called lipidomics, presents a number of opportunities not only for understanding the cellular processes in health and disease but also in enabling personalized medicine. Lipidomics in its most advanced form is able to quantify hundreds of different molecular lipid species with various structural and functional roles. Unraveling this complexity will improve our understanding of diseases such as atherosclerosis at a level of detail not attainable with classical analytical methods. Improved patient selection, biomarkers for gauging treatment efficacy and safety, and translational models will be facilitated by the lipidomic deliverables. Importantly, lipid-based biomarkers and targets should lead the way as we progress toward more specialized therapeutics

B-MYB Is Essential for Normal Cell Cycle Progression and Chromosomal Stability of Embryonic Stem Cells

Background: The transcription factor B-Myb is present in all proliferating cells, and in mice engineered to remove this gene, embryos die in utero just after implantation due to inner cell mass defects. This lethal phenotype has generally been attributed to a proliferation defect in the cell cycle phase of G1. Methodology/Principal Findings: In the present study, we show that the major cell cycle defect in murine embryonic stem (mES) cells occurs in G2/M. Specifically, knockdown of B-Myb by short-hairpin RNAs results in delayed transit through G2/M, severe mitotic spindle and centrosome defects, and in polyploidy. Moreover, many euploid mES cells that are transiently deficient in B-Myb become aneuploid and can no longer be considered viable. Knockdown of B-Myb in mES cells also decreases Oct4 RNA and protein abundance, while over-expression of B-MYB modestly up-regulates pou5f1 gene expression. The coordinated changes in B-Myb and Oct4 expression are due, at least partly, to the ability of B-Myb to directly modulate pou5f1 gene promoter activity in vitro. Ultimately, the loss of B-Myb and associated loss of Oct4 lead to an increase in early markers of differentiation prior to the activation of caspase-mediated programmed cell death. Conclusions/Significance: Appropriate B-Myb expression is critical to the maintenance of chromosomally stable and pluripotent ES cells, but its absence promotes chromosomal instability that results in either aneuploidy or differentiation-associated cell death

Genetic association study of QT interval highlights role for calcium signaling pathways in myocardial repolarization.

The QT interval, an electrocardiographic measure reflecting myocardial repolarization, is a heritable trait. QT prolongation is a risk factor for ventricular arrhythmias and sudden cardiac death (SCD) and could indicate the presence of the potentially lethal mendelian long-QT syndrome (LQTS). Using a genome-wide association and replication study in up to 100,000 individuals, we identified 35 common variant loci associated with QT interval that collectively explain ∼8-10% of QT-interval variation and highlight the importance of calcium regulation in myocardial repolarization. Rare variant analysis of 6 new QT interval-associated loci in 298 unrelated probands with LQTS identified coding variants not found in controls but of uncertain causality and therefore requiring validation. Several newly identified loci encode proteins that physically interact with other recognized repolarization proteins. Our integration of common variant association, expression and orthogonal protein-protein interaction screens provides new insights into cardiac electrophysiology and identifies new candidate genes for ventricular arrhythmias, LQTS and SCD

52 Genetic Loci Influencing Myocardial Mass.

BACKGROUND: Myocardial mass is a key determinant of cardiac muscle function and hypertrophy. Myocardial depolarization leading to cardiac muscle contraction is reflected by the amplitude and duration of the QRS complex on the electrocardiogram (ECG). Abnormal QRS amplitude or duration reflect changes in myocardial mass and conduction, and are associated with increased risk of heart failure and death. OBJECTIVES: This meta-analysis sought to gain insights into the genetic determinants of myocardial mass. METHODS: We carried out a genome-wide association meta-analysis of 4 QRS traits in up to 73,518 individuals of European ancestry, followed by extensive biological and functional assessment. RESULTS: We identified 52 genomic loci, of which 32 are novel, that are reliably associated with 1 or more QRS phenotypes at p < 1 × 10(-8). These loci are enriched in regions of open chromatin, histone modifications, and transcription factor binding, suggesting that they represent regions of the genome that are actively transcribed in the human heart. Pathway analyses provided evidence that these loci play a role in cardiac hypertrophy. We further highlighted 67 candidate genes at the identified loci that are preferentially expressed in cardiac tissue and associated with cardiac abnormalities in Drosophila melanogaster and Mus musculus. We validated the regulatory function of a novel variant in the SCN5A/SCN10A locus in vitro and in vivo. CONCLUSIONS: Taken together, our findings provide new insights into genes and biological pathways controlling myocardial mass and may help identify novel therapeutic targets