Mechanistic and structural basis for the actions of the antibacterial gepotidacin against Staphylococcus aureus gyrase

Gepotidacin is a first-in-class triazaacenaphthylene novel bacterial topoisomerase inhibitor (NBTI). The compound has successfully completed phase II trials for the treatment of acute bacterial skin/skin structure infections and for the treatment of uncomplicated urogenital gonorrhea. It also displays robust in vitro activity against a range of wild-type and fluoroquinolone-resistant bacteria. Due to the clinical promise of gepotidacin, a detailed understanding of its interactions with its antibacterial targets is essential. Thus, we characterized the mechanism of action of gepotidacin against Staphylococcus aureus gyrase. Gepotidacin was a potent inhibitor of gyrase-catalyzed DNA supercoiling (IC50 ≈ 0.047 µM) and relaxation of positively supercoiled substrates (IC50 ≈ 0.6 µM). Unlike fluoroquinolones, which induce primarily double-stranded DNA breaks, gepotidacin induced high levels of gyrase-mediated single-stranded breaks. No double-stranded breaks were observed even at high gepotidacin concentration, long cleavage times, or in the presence of ATP. Moreover, gepotidacin suppressed the formation of double-stranded breaks. Gepotidacin formed gyrase-DNA cleavage complexes that were stable for >4 h. In vitro competition suggests that gyrase binding by gepotidacin and fluoroquinolones are mutually exclusive. Finally, we determined crystal structures of gepotidacin with the S. aureus gyrase core fusion truncate with nicked (2.31 Å resolution) or with intact (uncleaved) DNA (2.37 Å resolution). In both cases, a single gepotidacin molecule was bound midway between the two scissile DNA bonds and in a pocket between the two GyrA subunits. A comparison of the two structures demonstrates conformational flexibility within the central linker of gepotidacin, which may contribute to the activity of the compound

Building air leakage databases in energy conservation policies: analysis of selected initiatives in 4 European countries and the USA

Fulltext is available at http://tightvent.eu/wp-content/uploads/2012/02/TightVentReport03.pdfWe collected information on existing envelope air leakage databases from countries that are involved in the AIVC-TightVent project “Development and applications of building air leakage databases”. This document summarizes the information from five countries: Czech Republic, France, Germany, UK, and USA. Even though our summary is not exhaustive of all existing data on whole-building envelope air leakage, it provides an overview of recent efforts from a number of countries. There are many reasons why different countries are collecting these data. We will summarize their motivations, which drive some of the differences in the types of data being gathered and how the data are analysed. Detailed information from each country is provided at the end of this document in the form of tables

The Role of Translation Initiation Regulation in Haematopoiesis

Organisation of RNAs into functional subgroups that are translated in response to extrinsic and intrinsic factors underlines a relatively unexplored gene expression modulation that drives cell fate in the same manner as regulation of the transcriptome by transcription factors. Recent studies on the molecular mechanisms of inflammatory responses and haematological disorders indicate clearly that the regulation of mRNA translation at the level of translation initiation, mRNA stability, and protein isoform synthesis is implicated in the tight regulation of gene expression. This paper outlines how these posttranscriptional control mechanisms, including control at the level of translation initiation factors and the role of RNA binding proteins, affect hematopoiesis. The clinical relevance of these mechanisms in haematological disorders indicates clearly the potential therapeutic implications and the need of molecular tools that allow measurement at the level of translational control. Although the importance of miRNAs in translation control is well recognised and studied extensively, this paper will exclude detailed account of this level of control

An initial assessment of SMAP soil moisture retrievals using high-resolution model simulations and in situ observations

At the end of its first year of operation, we compare soil moisture retrievals from the Soil Moisture Active Passive (SMAP) mission to simulations from a land surface model with meteorological forcing downscaled from observations/reanalysis and in situ observations from sparse monitoring networks within continental United States (CONUS). The radar failure limits the duration of comparisons for the active and combined products (~3 months). Nevertheless, the passive product compares very well against in situ observations over CONUS. On average, SMAP compares to the in situ data even better than the land surface model and provides significant added value on top of the model and thus good potential for data assimilation. At large scale, SMAP is in good agreement with the model in most of CONUS with less-than-expected degradation over mountainous areas. Lower correlation between SMAP and the model is seen in the forested east CONUS and significantly lower over the Canadian boreal forests.United States. National Aeronautics and Space Administration (NNX14AH92G)United States. National Aeronautics and Space Administration (NNX13AI44G

Computing with quasiseparable matrices

International audienceThe class of quasiseparable matrices is defined by a pair of bounds, called the quasiseparable orders, on the ranks of the maximal sub-matrices entirely located in their strictly lower and upper triangular parts. These arise naturally in applications, as e.g. the inverse of band matrices, and are widely used for they admit structured representations allowing to compute with them in time linear in the dimension and quadratic with the quasiseparable order. We show, in this paper, the connection between the notion of quasisepa-rability and the rank profile matrix invariant, presented in [Dumas & al. ISSAC'15]. This allows us to propose an algorithm computing the quasiseparable orders (rL, rU) in time O(n^2 s^(ω−2)) where s = max(rL, rU) and ω the exponent of matrix multiplication. We then present two new structured representations, a binary tree of PLUQ decompositions, and the Bruhat generator, using respectively O(ns log n/s) and O(ns) field elements instead of O(ns^2) for the previously known generators. We present algorithms computing these representations in time O(n^2 s^(ω−2)). These representations allow a matrix-vector product in time linear in the size of their representation. Lastly we show how to multiply two such structured matrices in time O(n^2 s^(ω−2))

Thiophene antibacterials that allosterically stabilize DNA-cleavage complexes with DNA gyrase

A paucity of novel acting antibacterials is in development to treat the rising threat of antimicrobial resistance, particularly in Gram-negative hospital pathogens, which has led to renewed efforts in antibiotic drug discovery. Fluoroquinolones are broad-spectrum antibacterials that target DNA gyrase by stabilizing DNA-cleavage complexes, but their clinical utility has been compromised by resistance. We have identified a class of antibacterial thiophenes that target DNA gyrase with a unique mechanism of action and have activity against a range of bacterial pathogens, including strains resistant to fluoroquinolones. Although fluoroquinolones stabilize double-stranded DNA breaks, the antibacterial thiophenes stabilize gyrase-mediated DNA-cleavage complexes in either one DNA strand or both DNA strands. X-ray crystallography of DNA gyrase–DNA complexes shows the compounds binding to a protein pocket between the winged helix domain and topoisomerase-primase domain, remote from the DNA. Mutations of conserved residues around this pocket affect activity of the thiophene inhibitors, consistent with allosteric inhibition of DNA gyrase. This druggable pocket provides potentially complementary opportunities for targeting bacterial topoisomerases for antibiotic development

Physicochemical Characterization of Zeolites SSZ-26 and SSZ-33

Detailed characterization of zeolites SSZ-26 and SSZ-33 is presented and includes data from adsorption experiments, X-ray powder diffraction (XRD), electron diffraction (ED), and high-resolution electron microscopy (HREM). Simulated XRD patterns and HREM images of the proposed structures agree very well with all the experimental data. These zeolites are the first synthetic high-silica molecular sieves to contain a three-dimensional pore system comprised of intersecting 10- and 12-ring pores. The structures of SSZ-26 and SSZ-33 are compared to the structures of known zeolites, and the relationships between the geometry of the structure-directing agents used in the synthesis of SSZ-26 and the geometry of the pores are analyzed using energy minimization calculations. Several hypothetical zeolite structures are derived based on the proposed models for SSZ-26 and SSZ-33. SSZ-26 and SSZ-33 are the first examples of molecular sieves whose multidimensional pore systems have been formed by the purposeful design of their structure-directing agents

Physicochemical Characterization of Zeolites SSZ-26 and SSZ-33

Detailed characterization of zeolites SSZ-26 and SSZ-33 is presented and includes data from adsorption experiments, X-ray powder diffraction (XRD), electron diffraction (ED), and high-resolution electron microscopy (HREM). Simulated XRD patterns and HREM images of the proposed structures agree very well with all the experimental data. These zeolites are the first synthetic high-silica molecular sieves to contain a three-dimensional pore system comprised of intersecting 10- and 12-ring pores. The structures of SSZ-26 and SSZ-33 are compared to the structures of known zeolites, and the relationships between the geometry of the structure-directing agents used in the synthesis of SSZ-26 and the geometry of the pores are analyzed using energy minimization calculations. Several hypothetical zeolite structures are derived based on the proposed models for SSZ-26 and SSZ-33. SSZ-26 and SSZ-33 are the first examples of molecular sieves whose multidimensional pore systems have been formed by the purposeful design of their structure-directing agents

RASSF1A–LATS1 signalling stabilizes replication forks by restricting CDK2-mediated phosphorylation of BRCA2

Genomic instability is a key hallmark of cancer leading to tumour heterogeneity and therapeutic resistance. ​BRCA2 has a fundamental role in error-free DNA repair but also sustains genome integrity by promoting ​RAD51 nucleofilament formation at stalled replication forks. ​CDK2 phosphorylates ​BRCA2 (pS3291-​BRCA2) to limit stabilizing contacts with polymerized ​RAD51; however, how replication stress modulates ​CDK2 activity and whether loss of pS3291-​BRCA2 regulation results in genomic instability of tumours are not known. Here we demonstrate that the Hippo pathway kinase ​LATS1 interacts with ​CDK2 in response to genotoxic stress to constrain pS3291-​BRCA2 and support ​RAD51 nucleofilaments, thereby maintaining genomic fidelity during replication stalling. We also show that ​LATS1 forms part of an ​ATR-mediated response to replication stress that requires the tumour suppressor ​RASSF1A. Importantly, perturbation of the ​ATR–​RASSF1A–​LATS1 signalling axis leads to genomic defects associated with loss of ​BRCA2 function and contributes to genomic instability and ‘BRCA-ness’ in lung cancers

Fibulin 1 is downregulated through promoter hypermethylation in gastric cancer

Tumour suppressor genes (TSGs) were frequently inactivated through promoter hypermethylation in gastric carcinoma as well as pre-malignant gastric lesions, suggesting that promoter hypermethylation can be used as a marker to define novel TSGs and also biomarkers for early detection of gastric cancer. In an effort to search for such genes aberrantly methylated in gastric cancer development, fibulin 1 (FBLN1) was found as a candidate TSG epigenetically downregulated in gastric cancer. FBLN1 expression was downregulated in all of gastric cancer cell lines used (100%, 7 out of 7) and the primary gastric carcinoma tissues (84%, 86 out of 102) and significantly restored after pharmacological demethylation. Hypermethylation of the FBLN1 promoter was frequently (71%, 5 out of 7) detected in gastric cancer cell lines and primary gastric carcinoma tissues. Ectopic expression of FBLN1 led to the growth inhibition of gastric cancer cells through the induction of apoptosis. In summary, FBLN1 was identified as a novel candidate TSG epigenetically downregulated in gastric cancer