Crystal structure of the Pseudomonas aeruginosa BEL-1 extended-spectrum β-lactamase and its complexes with moxalactam and imipenem

BEL-1 is an acquired class A extended-spectrum β-lactamase (ESBL) found in Pseudomonas aeruginosa clinical isolates from Belgium which is divergent from other ESBLs (maximum identity of 54% with GES-type enzymes). This enzyme is efficiently inhibited by clavulanate, imipenem, and moxalactam. Crystals of BEL-1 were obtained at pH 5.6, and the structure of native BEL-1 was determined from orthorhombic and monoclinic crystal forms at 1.60-Å and 1.48-Å resolution, respectively. By soaking native BEL-1 crystals, complexes with imipenem (monoclinic form, 1.79-Å resolution) and moxalactam (orthorhombic form, 1.85-Å resolution) were also obtained. In the acyl-enzyme complexes, imipenem and moxalactam differ by the position of the α- substituent and of the carbonyl oxygen (in or out of the oxyanion hole). More surprisingly, the Ω-loop, which includes the catalytically relevant residue Glu166, was found in different conformations in the various subunits, resulting in the Glu166 side chain being rotated out of the active site or even in displacement of its Cα atom up to approximately 10 Å. A BEL-1 variant showing the single Leu162Phe substitution (BEL- 2) confers a higher level of resistance to CAZ, CTX, and FEP and shows significantly lower Km values than BEL-1, especially with oxyiminocephalosporins. BEL-1 Leu162 is located at the beginning of the Ω-loop and is surrounded by Phe72, Leu139, and Leu148 (contact distances, 3.5 to 3.9 Å). This small hydrophobic cavity could not reasonably accommodate the bulkier Phe162 found in BEL-2 without altering neighboring residues or the Ω-loop itself, thus likely causing an important alteration of the enzyme kinetic properties

Application of a general discrete adjoint method for draft tube optimization

ABSTRACT: Automatic optimization is becoming increasingly important in turbomachinery design to improve the performance of machine components and Evolutionary Algorithms (EAs) play a very important role in this task. The main drawback of EAs is the large number of evaluations that are required to obtain an "optimal" result. Consequently, in order to keep the computational time in an affordable frame for design purposes, either the mesh size has to be limited, thus reducing the resolution of the flow phenomena, or the number of free parameters must be kept small. Adjoint optimization does not suffer from these restrictions, i.e. the optimization time is not affected by the number of parameters. The computational effort for the adjoint method scales only with the grid size and is usually in the range of two times the CFD simulation alone. In this paper, a discrete adjoint method based on a coupled pressure based RANS solver is presented and applied to draft tube optimization. The adjoint solver is general and can therefore deal with any turbulence model supported by the CFD solver as well as any boundary condition, including mixing planes and mesh interfaces needed for multi-stage simulations. Furthermore, there is no restriction on the choice of objective function. The adjoint method is first applied to a baseline draft tube geometry and then again to its EA optimized geometry where the objective function was the minimization of losses in the draft tube. To reduce the complexity for this proof of concept but still including multiple operating points in the optimization, only peak efficiency and full-load were optimized simultaneously. The adjoint optimization can significantly improve the draft tube performance in both cases (baseline and EA optimization). The interplay between local and global optimization seems to be a promising strategy to find optimal geometries for multi-operating point/multi-objective optimization and will be further investigated in subsequent research

The Role of PIXE and XRF in Heritage Science: The INFN-CHNet LABEC Experience

Analytical techniques play a fundamental role in heritage science. Among them, Particle Induced X-ray Emission (PIXE) and X-ray Fluorescence (XRF) techniques are widely used in many laboratories for elemental composition analysis. Although they are well-established, a strong effort is put on their upgrade, making them suitable for more and more applications. Over the years, at the INFN-LABEC (the laboratory of nuclear techniques for the environment and cultural heritage of the Italian National Institute of Nuclear Physics), the INFN-CHNet group, the network devoted to cultural heritage, has carried out many technological improvements to the PIXE and XRF set-ups for the analysis of works of art and archaeological finds. Among the many, we recall here the scanning external microbeam facility at the TANDEM accelerator and the MA-XRF scanner. The two instruments have shown complementary features: the former permits quantitative analysis of elements heavier than sodium, which is not possible with the latter in most of the case studies. On the contrary, the scanner has the undeniable advantage of portability, allowing it to work in situ. In this framework of technological developments in heritage science, INFN, CERN, and OPD are jointly carrying on the MACHINA (Movable Accelerator for Cultural Heritage In-situ Non-destructive Analysis) project for on-site Ion Beam Analysis (IBA) studies on cultural heritage

SAR Studies Leading to the Identification of a Novel Series of Metallo-β-lactamase Inhibitors for the Treatment of Carbapenem-Resistant Enterobacteriaceae Infections That Display Efficacy in an Animal Infection Model

The clinical effectiveness of carbapenem antibiotics such as meropenem is becoming increasingly compromised by the spread of both metallo-β-lactamase (MBL) and serine-β-lactamase (SBL) enzymes on mobile genetic elements, stimulating research to find new β-lactamase inhibitors to be used in conjunction with carbapenems and other β-lactam antibiotics. Herein, we describe our initial exploration of a novel chemical series of metallo-β-lactamase inhibitors, from concept to efficacy, in a survival model using an advanced tool compound (ANT431) in conjunction with meropenem

Profiling of Flavonol Derivatives for the Development of Antitrypanosomatidic Drugs

Flavonoids represent a potential source of new antitrypanosomatidic leads. Starting from a library of natural products, we combined target-based screening on pteridine reductase 1 with phenotypic screening on Trypanosoma brucei for hit identification. Flavonols were identified as hits, and a library of 16 derivatives was synthesized. Twelve compounds showed EC50 values against T. brucei below 10 \u3bcM. Four X-ray crystal structures and docking studies explained the observed structure-activity relationships. Compound 2 (3,6-dihydroxy-2-(3-hydroxyphenyl)-4H-chromen-4-one) was selected for pharmacokinetic studies. Encapsulation of compound 2 in PLGA nanoparticles or cyclodextrins resulted in lower in vitro toxicity when compared to the free compound. Combination studies with methotrexate revealed that compound 13 (3-hydroxy-6-methoxy-2-(4-methoxyphenyl)-4H-chromen-4-one) has the highest synergistic effect at concentration of 1.3 \u3bcM, 11.7-fold dose reduction index and no toxicity toward host cells. Our results provide the basis for further chemical modifications aimed at identifying novel antitrypanosomatidic agents showing higher potency toward PTR1 and increased metabolic stability

Exploiting the 2-Amino-1,3,4-thiadiazole Scaffold To Inhibit <i>Trypanosoma brucei </i>Pteridine Reductase in Support of Early-Stage Drug Discovery

Pteridine reductase-1 (PTR1) is a promising drug target for the treatment of trypanosomiasis. We investigated the potential of a previously identified class of thiadiazole inhibitors of Leishmania major PTR1 for activity against Trypanosoma brucei (Tb). We solved crystal structures of several TbPTR1-inhibitor complexes to guide the structure-based design of new thiadiazole derivatives. Subsequent synthesis and enzyme- and cell-based assays confirm new, mid-micromolar inhibitors of TbPTR1 with low toxicity. In particular, compound 4m, a biphenyl-thiadiazole-2,5-diamine with IC50 = 16 μM, was able to potentiate the antitrypanosomal activity of the dihydrofolate reductase inhibitor methotrexate (MTX) with a 4.1-fold decrease of the EC50 value. In addition, the antiparasitic activity of the combination of 4m and MTX was reversed by addition of folic acid. By adopting an efficient hit discovery platform, we demonstrate, using the 2-amino-1,3,4-thiadiazole scaffold, how a promising tool for the development of anti-T. brucei agents can be obtained

What influences media coverage? African countries in major Italian newspapers: 1996-2015

The mass media play a key role on the public and policy agenda, especially for events and issues regarding foreign countries. However, it is not clear the rationale involved in the media coverage. This paper studies the references to African countries in two major Italian newspapers between 1996 and 2015. A panel analysis shows that the macroeconomic conditions and the occurrence of coups d'état do not exert a decisive impact on the mentions of a country, while the countries with the lowest life expectancy are more frequently in the news