Coupled system thermal Hydraulics/CFD models: General guidelines and applications to heavy liquid metals

This work aims to review the general guidelines to be adopted to perform coupled System Thermal Hydraulics (STH)/CFD calculations. The coupled analysis is often required when complex phenomena characterized by different characteristic time and length scales are investigated. Indeed, by STH/CFD coupling the main drawbacks of both stand-alone codes are overcome, reducing the computational cost and providing more realistic solutions. A review of several works available in literature and involving different coupling approaches, codes, time-advancing schemes and application fields is given. Besides STH/CFD coupling techniques, spatial domains and numerical schemes are analysed in detail. A brief description of applications to heavy liquid metal systems is also reported; lessons drawn in the frame of these and other works are then considered in order to develop a set of good practice guidelines for coupled STH/CFD applications

RELAP5/SIMMER-III code coupling development for PbLi-water interaction

A major safety issue in the Water-Cooled Lead-Lithium Breeding Blanket (WCLL-BB) system foreseen for fusion reactor is the interaction concerning the primary coolant (water) and the neutron multiplier (PbLi), due to a hypothetical tube rupture in the coolant circuit. This scenario involves an exothermic chemical reaction between PbLi and water with the production of hydrogen, in addition to critical interactions in a complex multiphase system in non-thermal equilibrium. In recent years the PbLi/water reaction was successfully implemented in the SIMMER-III code and validated against data from the LIFUS5/Mod3 experimental campaign. However, due to limitations of SIMMER-III, this work was restricted to the prediction of the phenomena inside the vessel, neglecting the simulation of the injection line. Nevertheless, since the injection line may actually have an important effect on the development of the transient, the simulation of the whole facility would be highly desirable. Indeed, the University of Pisa recently developed a coupling methodology between the SIMMER-III and RELAP5/Mod3.3 codes and applied it to simple single-phase cases. In this paper the complete simulation of the LIFUS5/Mod3 facility is presented, with the injection line modelled through RELAP5. Furthermore, all the complex aspects of the phenomena inside the reaction tank were included: the multiphase system and the interaction between water and PbLi with the chemical reaction and the production of hydrogen were modelled by SIMMER. Preliminary results are presented, showing that the coupling methodology can be effectively employed for the prediction of the chemical and thermal-hydraulic behaviour of complex loop experimental facilities

A standard numbering scheme for class C β-lactamases

Unlike classes A and B, a standardized amino acid numbering scheme has not been proposed for the class C (AmpC) β-lactamases, which complicates communication in the field. Here, we propose a scheme developed through a collaborative approach that considers both sequence and structure, preserves traditional numbering of catalytically important residues (Ser64, Lys67, Tyr150, and Lys315), is adaptable to new variants or enzymes yet to be discovered, and includes a variation for genetic and epidemiological applications

Metal Ion Binding and Coordination Geometry for Wild Type and Mutants of Metallo-Beta -Lactamase from Bacillus Cereus 569/H/9 (Bcii): A Combined Thermodynamic, Kinetic, and Spectroscopic Approach

peer reviewedOne high affinity (nm) and one low affinity (microM) macroscopic dissociation constant for the binding of metal ions were found for the wild-type metallo-beta-lactamase from Bacillus cereus as well as six single-site mutants in which all ligands in the two metal binding sites were altered. Surprisingly, the mutations did not cause a specific alteration of the affinity of metal ions for the sole modified binding site as determined by extended x-ray absorption fine structure (EXAFS) and perturbed angular correlation of gamma-rays spectroscopy, respectively. Also UV-visible absorption spectra for the mono-cobalt enzymes clearly contain contributions from both metal sites. The observations of the very similar microscopic dissociation constants of both binding sites in contrast to the significantly differing macroscopic dissociation constants inevitably led to the conclusion that binding to the two metal sites exhibits negative cooperativity. The slow association rates for forming the binuclear enzyme determined by stopped-flow fluorescence measurements suggested that fast metal exchange between the two sites for the mononuclear enzyme hinders the binding of a second metal ion. EXAFS spectroscopy of the mono- and di-zinc wild type enzymes and two di-zinc mutants provide a definition of the metal ion environments, which is compared with the available x-ray crystallographic data

Structural basis of metallo-β-lactamase, serine-β-lactamase and penicillin-binding protein inhibition by cyclic boronates

β-Lactamases enable resistance to almost all β-lactam antibiotics. Pioneering work revealed that acyclic boronic acids can act as ‘transition state analogue’ inhibitors of nucleophilic serine enzymes, including serine-β-lactamases. Here we report biochemical and biophysical analyses revealing that cyclic boronates potently inhibit both nucleophilic serine and zinc-dependent β-lactamases by a mechanism involving mimicking of the common tetrahedral intermediate. Cyclic boronates also potently inhibit the non-essential penicillin-binding protein PBP 5 by the same mechanism of action. The results open the way for development of dual action inhibitors effective against both serine- and metallo-β-lactamases, and which could also have antimicrobial activity through inhibition of PBPs

Enumerating Pathways of Proton Abstraction Based on a Spatial and Electrostatic Analysis of Residues in the Catalytic Site

The pathways of proton abstraction (PA), a key aspect of most catalytic reactions, is often controversial and highly debated. Ultrahigh-resolution diffraction studies, molecular dynamics, quantum mechanics and molecular mechanic simulations are often adopted to gain insights in the PA mechanisms in enzymes. These methods require expertise and effort to setup and can be computationally intensive. We present a push button methodology – Proton abstraction Simulation (PRISM) – to enumerate the possible pathways of PA in a protein with known 3D structure based on the spatial and electrostatic properties of residues in the proximity of a given nucleophilic residue. Proton movements are evaluated in the vicinity of this nucleophilic residue based on distances, potential differences, spatial channels and characteristics of the individual residues (polarity, acidic, basic, etc). Modulating these parameters eliminates their empirical nature and also might reveal pathways that originate from conformational changes. We have validated our method using serine proteases and concurred with the dichotomy in PA in Class A β-lactamases, both of which are hydrolases. The PA mechanism in a transferase has also been corroborated. The source code is made available at www.sanchak.com/prism

Hidden heterochromatin: Characterization in the Rodentia species Cricetus cricetus, Peromyscus eremicus (Cricetidae) and Praomys tullbergi (Muridae)

The use of in situ restriction endonuclease (RE) (which cleaves DNA at specific sequences) digestion has proven to be a useful technique in improving the dissection of constitutive heterochromatin (CH), and in the understanding of the CH evolution in different genomes. In the present work we describe in detail the CH of the three Rodentia species, Cricetus cricetus, Peromyscus eremicus (family Cricetidae) and Praomys tullbergi (family Muridae) using a panel of seven REs followed by C-banding. Comparison of the amount, distribution and molecular nature of C-positive heterochromatin revealed molecular heterogeneity in the heterochromatin of the three species. The large number of subclasses of CH identified in Praomys tullbergi chromosomes indicated that the karyotype of this species is the more derived when compared with the other two genomes analyzed, probably originated by a great number of complex chromosomal rearrangements. The high level of sequence heterogeneity identified in the CH of the three genomes suggests the coexistence of different satellite DNA families, or variants of these families in these genomes

One origin for metallo-β-lactamase activity, or two? An investigation assessing a diverse set of reconstructed ancestral sequences based on a sample of phylogenetic trees

This work was supported by BBSRC (grant BB/F016778/1)Bacteria use metallo-β-lactamase enzymes to hydrolyse lactam rings found in many antibiotics, rendering them ineffective. Metallo-β-lactamase activity is thought to be polyphyletic, having arisen on more than one occasion within a single functionally diverse homologous superfamily. Since discovery of multiple origins of enzymatic activity conferring antibiotic resistance has broad implications for the continued clinical use of antibiotics, we test the hypothesis of polyphyly further; if lactamase function has arisen twice independently, the most recent common ancestor (MRCA) is not expected to possess lactam-hydrolysing activity. Two major problems present themselves. Firstly, even with a perfectly known phylogeny, ancestral sequence reconstruction is error prone. Secondly, the phylogeny is not known, and in fact reconstructing a single, unambiguous phylogeny for the superfamily has proven impossible. To obtain a more statistical view of the strength of evidence for or against MRCA lactamase function, we reconstructed a sample of 98 MRCAs of the metallo-β-lactamases, each based on a different tree in a bootstrap sample of reconstructed phylogenies. InterPro sequence signatures and homology modelling were then used to assess our sample of MRCAs for lactamase functionality. Only 5 % of these models conform to our criteria for metallo-β-lactamase functionality, suggesting that the ancestor was unlikely to have been a metallo-β-lactamase. On the other hand, given that ancestral proteins may have had metallo-β-lactamase functionality with variation in sequence and structural properties compared with extant enzymes, our criteria are conservative, estimating a lower bound of evidence for metallo-β-lactamase functionality but not an upper bound.Publisher PDFPeer reviewe

A survey of the kinetic parameters of class C beta-lactamases. Penicillins.

The interaction between six class C beta-lactamases and various penicillins has been studied. All the enzymes behaved in a very uniform manner. Benzylpenicillin exhibited relatively low kcat. values (14-75 s-1) but low values of Km resulted in high catalytic efficiencies [kcat./Km = 10 X 10(6)-75 X 10(6) M-1.s-1]. The kcat. values for ampicillin were 10-100-fold lower. Carbenicillin, oxacillin cloxacillin and methicillin were very poor substrates, exhibiting kcat. values between 1 x 10(-3) and 0.1 s-1. The Km values were correspondingly small. It could safely be hypothesized that, with all the tested substrates, deacylation was rate-limiting, resulting in acyl-enzyme accumulation