ALDB: Debugging Alloy Models of Behavioural Requirements

© 2020 IEEE. Personal use of this material is permitted. Permission from IEEE must be obtained for all other uses, in any current or future media, including reprinting/republishing this material for advertising or promotional purposes, creating new collective works, for resale or redistribution to servers or lists, or reuse of any copyrighted component of this work in other works.Declarative modelling languages, such as Alloy, are becoming popular for describing behavioural requirements very early in system development because automated analysis of these models provides valuable feedback. Typically, these languages are supported by constraint solvers (SAT, SMT) for providing instances or model checking properties. However, a user can quickly find simple bugs and gain confidence in their model by concretely simulating steps of the transition system. We present ALDB: a debugger for models of transition systems written in the Alloy language. It provides a familiar debugging interface to walk around in the behaviour of the model, enabling users to quickly explore scenarios, find errors via concrete simulation, and incrementally build up to bounded model checking.Natural Sciences and Engineering Research Council of Canada

Anakinra in Heart Failure: A Systematic Review and Meta-Analysis of Randomized Controlled Trials

Background: Heart failure (HF) has become increasingly difficult to manage given its increasing incidence. Despite the availability of novel treatment target relieving inhibition and congestions for neurohormonal activation, heart failure is one of leading health conditions associated with high hospitalization and readmission rates, resulting in poor quality of life. In light of this, this article serves to demonstrate the effect of anakinra as one of the treatment paradigms for HF to explore the need for advanced novel interventions. Methods: We conducted a search in five electronic databases, including Embase, MEDLINE, Cochrane, Scopus, and PubMed, for RCTs (randomized controlled trials) evaluating the effects of anakinra against placebo in HF. Meta-analysis was performed using RevMan version 5.4. Results: Eight RCTs were obtained and included for analysis in this study. The results demonstrate that anakinra significantly reduces the levels of CRP (C-reactive protein), with significant difference between anakinra- and placebo-treated groups. Analyses also show that CRP failed to cause an improvement in peak oxygen consumption and ventilatory efficiency. Additionally, the treatment-related adverse events were insignificant. Some considerable limitations are that the same set of researchers were involved in most of the studies; hence, more independent studies need to be encouraged. Conclusion: Anakinra was associated with a reduction in CRP levels, indicating some anti-inflammatory effects but no effect on function, exercise capacity, and adverse effects

Dengue Virus Nonstructural Protein 5 (NS5) Assembles into a Dimer with a Unique Methyltransferase and Polymerase Interface

<div><p>Flavivirus nonstructural protein 5 (NS5) consists of methyltransferase (MTase) and RNA-dependent RNA polymerase (RdRp) domains, which catalyze 5’-RNA capping/methylation and RNA synthesis, respectively, during viral genome replication. Although the crystal structure of flavivirus NS5 is known, no data about the quaternary organization of the functional enzyme are available. We report the crystal structure of dengue virus full-length NS5, where eight molecules of NS5 are arranged as four independent dimers in the crystallographic asymmetric unit. The relative orientation of each monomer within the dimer, as well as the orientations of the MTase and RdRp domains within each monomer, is conserved, suggesting that these structural arrangements represent the biologically relevant conformation and assembly of this multi-functional enzyme. Essential interactions between MTase and RdRp domains are maintained in the NS5 dimer via inter-molecular interactions, providing evidence that flavivirus NS5 can adopt multiple conformations while preserving necessary interactions between the MTase and RdRp domains. Furthermore, many NS5 residues that reduce viral replication are located at either the inter-domain interface within a monomer or at the inter-molecular interface within the dimer. Hence the X-ray structure of NS5 presented here suggests that MTase and RdRp activities could be coordinated as a dimer during viral genome replication.</p></div

NS5 dimer interactions.

<p><b>(A)</b> Type I dimer. All eight NS5 monomers are involved in type I dimer interaction (AB, CD, EF, and GH). One monomer is colored as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005451#ppat.1005451.g002" target="_blank">Fig 2A</a> and the other is in gray. Residues of potential physiological significance are shown as magenta (MTase) or gold (RdRp) spheres, and labeled with corresponding one-letter residue codes. The close-up view of the type I dimer interface is shown as sticks (right), and hydrogen bonds are indicated by dashed lines. <b>(B)</b> Type II dimer. The type II dimers are observed only between monomers A and F and between D and G. The close-up view of the C-terminal residues involved in the dimer interface is shown as sticks (right).</p

Serotype–specific interactions in DENV NS5.

<p>(A) Immunofluorescence assay. DENV2 RNA encoding the wild-type (DENV2) NS5, a chimera NS5 containing DENV4 MTase, or DENV4 NS5 were transfected into BHK-21 cells, and viral replication was visualized by immunofluorescence assay using anti NS1 antibodies. (B) Serotype-specific NS5 residues. Serotype-specific residues (boxed) are conserved within one DENV serotype, but not conserved across serotypes. Please see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005451#sec012" target="_blank">Methods</a> section for details of sequence alignment and determination of serotype-specific residues. (C) Serotype-specific residues for at least two serotypes of DENV NS5 are mapped on the 3-D structure of the DENV3 NS5 dimer. One monomer is colored in cyan (MTase domain) and yellow (RdRp domain), and the other in gray. Serotype-specific residues are colored in red and labeled. The NLS (residues 369–406) is colored in blue.</p

DENV NS5 monomer.

<p><b>(A)</b> Overall fold of the DENV NS5 monomer. The ribbon diagram of the NS5 monomer is shown looking through the canonical right hand configuration (left) and through the top of the RdRp (right). The MTase domain is shown in cyan, and the RdRp domain is colored by region (thumb, red; palm, green; fingers, blue; domain linker, orange; priming loop, yellow). SAH is shown as a space-filling model and colored by atom type. Two zinc ions are shown as gray spheres. Schematic of the NS5 domains is shown below. <b>(B)</b> Polymerase activities of NS5 proteins. Polymerase activities of NS5 proteins were measured using a subgenomic RNA as a template, as previously described [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005451#ppat.1005451.ref027" target="_blank">27</a>]. Lanes: 1, no polymerase; 2, NS5 RdRp domain; 3, wild-type NS5; 4, NS5-Δ2; 5, NS5-Δ6. Please see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005451#sec012" target="_blank">Materials and Methods</a> for specific deletions. (C) Comparison of RdRp domains of DENV and JEV NS5. The RdRp domains of the two DENV (the current structure and PDB code 4V0Q) and JEV (PDB code 4K6M) are compared. The previously disordered regions including motif F (green), motif G (blue), linker (orange), and the C-terminus (red) are labeled. Since none of the DENV NS5 monomers included both motif G and the C-terminal helix, we created a model of the monomer by grafting motif G from monomer C onto monomer G, which included the C-terminal helix; the resulting model thus has both motif G and the C-terminus. Motif F in DENV NS5 is disordered and shown as a dotted line. The priming loop is shown in purple. <b>(D)</b> Stereoview of the linker between the MTase and RdRp domains. The residues 260–272 are shown in orange, and the final 2<i>F</i>_<i>o</i>-<i>F</i>_<i>c</i> omit map for the linker region is shown as a blue mesh contoured at 1.0 σ. MTase and RdRp residues that interact with the linker are colored as in (A). Hydrogen bonds are indicated by dashed lines. <b>(E)</b> Movement of the thumb subdomain in flavivirus NS5. The RdRp domain of DENV NS5 (chain B, blue) was aligned with the JEV RdRp domain (orange) by fingers and palm subdomains (rmsd = 0.56 Å for 333 Cα atoms). The thumb subdomain of DENV NS5 is rotated by 7° around W700 (hinge residue, blue sphere) compared to the JEV thumb subdomain.</p

Comparison of DENV and JEV NS5 structures.

<p><b>(A)</b> Superposition of DENV and JEV NS5. DENV and JEV NS5 (PDB code 4K6M) are aligned by the MTase domains only (rmsd of 0.99 Å for 253 Cα atoms). The DENV NS5 structure is colored as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005451#ppat.1005451.g002" target="_blank">Fig 2A</a>, and the JEV NS5 is in gray. With the MTase domains superimposed, the RdRp domains of DENV and JEV NS5 are related by a rotation of 102° and a ~5Å translation along the linker region (residues 262–272). A close-up view of the boxed area is shown in (B). <b>(B)</b> The linker regions in DENV and JEV NS5. Residues comprising the linker regions of DENV and JEV NS5 are shown in orange and gray, respectively. Both structures use the ²⁶⁰GTR²⁶² pivot, following which the two structures diverge. The dotted line indicates the break in the JEV NS5 linker. <b>(C)</b> Comparison of DENV and JEV NS5 structures. To emphasize the relative orientations of the MTase and RdRp domains, SAH molecules that occupy an identical binding site in both structures are shown in yellow. P113, P115 (or L115 in JEV NS5), and W121 in the MTase are implicated in viral replication and shown in magenta spheres in ribbon diagrams or magenta surfaces in surface representations. These residues in DENV NS5 are located in the dimer interface, while those in JEV NS5 are located in the domain interface.</p

NS5 monomer and dimer models in the replication complex.

<p><b>(A)</b> Schematics of the NS5 monomer and dimer models. Two conformations of the MTase relative to the RdRp observed in DENV and JEV NS5 structures in the monomer model are indicated by black and gray lines. The template entry and dsRNA product exit sites on the RdRp are indicated by arrows. The MTase active site is represented by a concave region. <b>(B)</b> DENV NS5 dimer with bound RNA model. To show molecular boundaries, one monomer is shown as a ribbon diagram, and the other as a molecular surface. The MTase active site is indicated by the bound SAH (yellow) and a short RNA (orange). The single-stranded RNA was modeled in by superposition of the MTase domain in our structure and the isolated MTase domain-RNA complex (PDB code 2XBM) [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005451#ppat.1005451.ref045" target="_blank">45</a>].</p