Dissecting the maturation steps of the lasso peptide microcin J25 in vitro

Microcin J25 is the archetype of a growing class of bacterial ribosomal peptides possessing a knotted topology (lasso peptides). It consists of an eight-residue macrolactam ring through which the C-terminal tail is threaded. It is biosynthesized as a precursor that is processed by two maturation enzymes (McjB/McjC). Insights into the mechanism of microcin J25 biosynthesis have been provided previously by mutagenesis of the precursor peptide in vivo. In this study we have demonstrated distinct functions of McjB and McjC in vitro for the first time, based on the detection of reaction intermediates. McjB was characterized as a new ATP-dependent cysteine protease, whereas McjC was confirmed to be a lactam synthetase. The two enzymes were functionally interdependent, likely forming a structural complex. Their substrate preference was directly investigated with the aid of mutated precursor peptides. Depending on the substitutions, microcin J25 variants with either a lasso or branched-cyclic topology could be generated in vitro.     

Construction of a single polypeptide that matures and exports the lasso peptide microcin J25

Roped in: The lasso peptide microcin J25 (MccJ25) is matured by two enzymes and is exported by a putative ABC transporter. We probed the function of the maturation enzymes using mutagenesis. We demonstrate that fusions of the enzymes with intervening linkers can produce MccJ25. Even a 151 kDa tripartite fusion between the ABC transporter and the two enzymes is capable of producing and exporting MccJ25.     

Sequence determinants governing the topology and biological activity of a lasso peptide, microcin J25

Microcin J25 is a potent antibacterial peptide produced by Escherichia coli AY25. It displays a lasso structure, which consists of a knot involving an N-terminal macrolactam ring through which the C-terminal tail is threaded and sterically trapped. In this study, we rationally designed and performed site-specific mutations in order to pinpoint the sequence determinants of the lasso topology. Structures of the resulting variants were analysed by a combination of methods (mass spectrometry, NMR spectroscopy, enzymatic digestion), and correlated to the antibacterial activity. The selected mutations resulted in the production of branched-cyclic or lasso variants. The C-terminal residues below the ring (Tyr20, Gly21) and the size of the macrolactam ring were revealed to be critical for both the lasso scaffold and bioactivity, while shortening the loop region (Tyr9-Ser18) or extending the C-terminal tail below the ring did not alter the lasso structure, but differentially affected the antibacterial activity. These results provide new insights for the bioengineering of antibacterial agents using a lasso peptide as template.     

Computational design of reduced metabolic networks

Cellular functions are based on thousands of chemical reactions and transport processes, most of them being catalysed and regulated by specific proteins. Systematic gene knockouts have provided evidence that this complex reaction network possesses considerable redundancy, that is, alternative routes exist along which signals and metabolic fluxes may be directed to accomplish an identical output behaviour. This property is of particular importance in cases where parts of the reaction network are transiently or permanently impaired, for example, due to an infection or genetic alterations. Here we present a computational concept to determine enzyme-reduced metabolic networks that are still sufficient to accomplish a given set of cellular functions. Our approach consists of defining an objective function that expresses the compromise that has to be made between successive reduction of the network by omission of enzymes and its decreasing thermodynamic and kinetic feasibility. Optimisation of this objective function results in a linear mixed-integer program. With increasing weight given to the reduction of the number of enzymes, the total flux in the network increases and some of the reactions have to proceed in thermodynamically unfavourable directions. The approach was applied to two metabolic schemes: the energy and redox metabolism of red blood cells and the carbon metabolism of Methylobacterium extorquens. For these two example networks, we determined various variants of reduced networks differing in the number and types of disabled enzymes and disconnected reactions. Using a comprehensive kinetic model of the erythrocyte metabolism, we assess the kinetic feasibility of enzyme-reduced subnetworks. The number of enzymes predicted to be indispensable amounts to 14 (out of 28) for the erythrocyte scheme and 13 (out of 77) for the bacterium scheme, the largest group of enzymes predicted to be simultaneously dispensable amounts to 3 and 37 for these two systems. Our approach might contribute to identifying potential target enzymes for rational drug design, to rationalising gene-expression profiles of metabolic enzymes and to designing synthetic networks with highly specialised metabolic functions.     

Computational design of structured chemical products

In chemical product design, the aim is to formulate a product with desired performance. Ingredients and internal product structure are two key drivers of product performance with direct impact on the mechanical, electrical, and thermal properties. Thus, there is a keen interest in elucidating the dependence of product performance on ingredients, structure, and the manufacturing process to form the structure. Design of product structure, particularly microstructure, is an intrinsically complex problem that involves different phases of different physicochemical properties, mass fraction, morphology, size distribution, and interconnectivity. Recently, computational methods have emerged that assist systematic microstructure quantification and prediction. The objective of this paper is to review these computational methods and to show how these methods as well as other developments in product design can work seamlessly in a proposed performance, ingredients, structure, and manufacturing process framework for the design of structured chemical products. It begins with the desired target properties and key ingredients. This is followed by computation for microstructure and then selection of processing steps to realize this microstructure. The framework is illustrated with the design of nanodielectric and die attach adhesive products.     

基于CFD的强化裂解炉管设计

通过计算流体力学（CFD）的方法,将丙烷裂解反应动力学与流动方程、能量方程耦合,考察了在普通裂解炉管中加装中空立交盘（hollow cross-disk,HCD）内构件对管内流动及裂解反应的影响。结果发现,HCD内构件通过壁面几何形状变化重布了流场结构,以合理的压力损失为代价产生径向速度,并诱导产生纵向涡剪切破坏边界层,强化了流体的湍动程度,降低热阻,提高了温度分布均匀性。相比于普通炉管,加入中空立交盘后,裂解管丙烷转化率提高7.24%,烯烃选择性提高3.67%,乙烯收率降低0.87%,但丙烯收率大幅上升16.50%,烯烃总收率上升6.94%。此外发现,纵向涡产生的径向流动促进了近壁区高温流体和管中心区相对低温流体的换位,流体温度最高下降了0.7℃;与普通炉管相比,新型裂解管出口处重组分浓度下降了28.33%,说明加入中空立交盘可防止近壁面高温区域过度裂解,有助于抑制结焦。在此基础上,结合模拟所得的场分布数据,定量分析了HCD强化传热和传质的机理,并就阻力损失和强化效果做出综合评价。     

Computational flow modelling and design of bubble column reactors

The present design practice of the bubble column reactors is still closer to an art than science because of the complexity of the fluid mechanics. In view of this, there have been continuous attempts to understand the complex three-dimensional turbulent two-phase flow. The present paper reviews the modelling efforts on the flow patterns published in the last 30 years with relatively more focus on the last 10 years. Over this period, there have been sustained efforts to improve our understanding of the governing equations of the change (equations of continuity and motion) for two-phase flows. Both Eulerian and Lagrangian approaches have been extensively used. The development has been mainly on three fronts: (i) formulation of interfacial forces (ii) closure problem for the eddy viscosity and (iii) modelling of the correlations arising out of Reynolds averaging procedure.As regards to interface force terms, the published literature has been critically analysed. The present status of our understanding of the drag force, virtual mass force and lift force has been presented. The physical significance of the various formulations has been brought out. The mechanism of the energy transfer from gas to liquid phase has been explained. The developments in closure problem have been most dramatic. The progress of the past 30 years has been reviewed with a focus on the past 10 years. The published literature has been critically analysed and chronology of development has been presented. The effort has been concentrated on cylindrical bubble columns where results on flow pattern could be extended to the design. The studies on transient flow pattern in two-dimensional columns have not been covered because the subject is still under development and the results cannot be extended to the design objective. The closure problem is intimately linked with the physics of turbulence.An attempt has been made to develop a complete correspondence between an operation of real column and the model simulation. Attention has been focused on the cylindrical bubble columns because of their widespread applications in the industry. The effects of the superficial gas velocity, column diameter and bubble slip velocity on the flow pattern have been examined. Extensive comparison has been presented between the predicted and the experimental velocity profiles.For the design of the bubble columns, the knowledge of various design parameters (such as pressure drop, rate of mixing, residence time distribution of both the phases, heat and mass transfer coefficients) is needed. For the estimation of these parameters, the prevailing procedures are largely empirical. The fundamental basis for the estimations is possible through the understanding of the detailed macro- and micro-flow patterns. This basic direction has been the subject of several publications, particularly during the last 5 years. All these studies have been critically analysed in the present review paper. A coherent and holistic approach has been presented on the modelling of fluid mechanics and design of bubble column reactors.Recommendations have been made for the future research in this area.     

J型烟囱的设计介绍

介绍为６．３ｔ／ｈ燃油锅炉配套的采用直接排出方式的Ｊ型烟囱．阐述了此烟囱的结构和高度设计的系列计算．     

Computational design of an endo-1,4-beta-xylanase ligand binding site

The field of computational protein design has experienced important recent success. However, the de novo computational design of high-affinity protein-ligand interfaces is still largely an open challenge. Using the Rosetta program, we attempted the in silico design of a high-affinity protein interface to a small peptide ligand. We chose the thermophilic endo-1,4-β-xylanase from Nonomuraea flexuosa as the protein scaffold on which to perform our designs. Over the course of the study, 12 proteins derived from this scaffold were produced and assayed for binding to the target ligand. Unfortunately, none of the designed proteins displayed evidence of high-affinity binding. Structural characterization of four designed proteins revealed that although the predicted structure of the protein model was highly accurate, this structural accuracy did not translate into accurate prediction of binding affinity. Crystallographic analyses indicate that the lack of binding affinity is possibly due to unaccounted for protein dynamics in the 'thumb' region of our design scaffold intrinsic to the family 11 β-xylanase fold. Further computational analysis revealed two specific, single amino acid substitutions responsible for an observed change in backbone conformation, and decreased dynamic stability of the catalytic cleft. These findings offer new insight into the dynamic and structural determinants of the β-xylanase proteins.     

Computational design of molecular properties: spotlight on accuracy and tuning

The Laboratory for Computational Molecular Design at ISIC devises original and accurate methodologies to establish, in silico, key structure-property relationships of large chemical systems with particular emphasis on those associated with pi-conjugated framework. Herein, we discuss two specific focuses of our activities: i) the development of accurate formalisms based on Kohn-Sham density functional theory to achieve quantitative results for the energies and geometries of extended systems featuring weak interactions and ii) the introduction of schemes to probe and tune the effect of intra- and intermolecular charge transfer on molecular properties. The proposed methodologies are ideally designed to tackle and resolve some of today's relevant aspects associated with the properties of pi-conjugated molecules, such as identifying relationships resulting in high stacking capacities, proposing more stable alternative topologies to large acenes, and analyzing the course of reaction involving assemblies of pi-conjugated molecules.     

Computational fluid dynamics‐based design of finned steam cracking reactors

The use of one‐dimensional reactor models to simulate industrial steam cracking reactors has been one of the main limiting factors for the development of new reactor designs and the evaluation of existing three‐dimensional (3‐D) reactor configurations. Therefore, a 3‐D computational fluid dynamics approach is proposed in which the detailed free‐radical chemistry is for the first time accounted for. As a demonstration case, the application of longitudinally and helicoidally finned tubes as steam cracking reactors was investigated under industrially relevant conditions. After experimental validation of the modeling approach, a comprehensive parametric study allowed to identify optimal values of the fin parameters, that is, fin height, number of fins, and helix angle to maximize heat transfer. Reactive simulations of an industrial Millisecond propane cracker were performed for four distinct finned reactors using a reaction network of 26 species and 203 elementary reactions. The start‐of‐run tube metal skin temperatures could be reduced by up to 50 K compared to conventionally applied tubular reactors when applying optimal fin parameters. Implementation of a validated coking model for light feedstocks shows that coking rates are reduced up to 50%. However, the increased friction and inner surface area lead to pressure drops higher by a factor from 1.22 to 1.66 causing minor but significant shifts in light olefin selectivity. For the optimized helicoidally finned reactor the ethene selectivity dropped, whereas propene and 1,3‐butadiene selectivity increased with a similar amount. The presented methodology can be applied in a straightforward way to other 3‐D reactor designs and can be extended to more complex feedstocks such as naphtha. © 2013 American Institute of Chemical Engineers AIChE J 60: 794–808, 2014     

23.5-25 16PR工程机械轮胎的优化设计

对23.5-25 16PR工程机械轮胎进行优化设计.优化设计轮胎的外直径为1 590 mm,断面宽为555 mm,行驶面宽度为510 mm,胎圈着合宽度为485 mm;胎体帘布层采用8层1870dtex/2锦纶帘布,缓冲层采用4层930dtex/2锦纶帘布,胎面基部胶厚度为9 mm;由罐式水胎硫化改为罐式胶囊硫化.优化后成品轮胎充气外缘尺寸和物理性能符合国家标准要求,轮胎装卸更为方便,外观质量明显提高,同时降低了成本.     

Computational design of molecularly imprinted polymer for direct detection of melamine in milk

A novel protocol for use of molecularly imprinted polymer (MIP) in analysis of melamine is presented. Design of polymer for melamine has been achieved using a combination of computational techniques and laboratory trials, the former greatly reducing the duration of the latter. The compatibility and concerted effect of monomers and solvents were also investigated and discussed. Two novel open-source tools were presented which are the online polymer calculator from mipdatabase.com and the application of the Gromacs modelling suite to determine the ideal stoichiometric ratio between template and functional monomer. The MIP binding was characterised for several structural analogues at 1–100 μM concentrations. The use of divinylbenzene (DVB) as cross-linking polymer and itaconic acid as functional monomer allowed synthesis of MIP with imprint factor (IF) of 2.25 for melamine. This polymer was used in high-performance liquid chromatography (HPLC) for the rapid detection of melamine in spiked milk samples with an experimental run taking 7–8 min. This approach demonstrated the power of virtual tools in accelerated design of MIPs for practical applications.     

Computational design of heterogeneous catalysts and gas separation materials for advanced chemical processing

Functional materials are widely used in chemical industry in order to reduce the process cost while simultaneously increase the product quality.Considering their significant effects,systematic methods for the optimal selection and design of materials are essential.The conventional synthesis-and-test method for materials development is inefficient and costly.Additionally,the performance of the resulting materials is usually limited by the designer’s expertise.During the past few decades,computational methods have been significantly developed and they now become a very important tool for the optimal design of functional materials for various chemical processes.This article selectively focuses on two important process functional materials,namely heterogeneous catalyst and gas separation agent.Theoretical methods and representative works for computational screening and design of these materials are reviewed.     

23.5-25 16PR宽基工程机械轮胎的优化设计

对23.5-25 16PR宽基工程机械轮胎进行了优化设计,通过采取减小轮胎外直径、断面宽和轮胎行驶面宽,增大轮胎行驶面弧度高,设计美观新颖的花纹形式,调整轮胎钢丝圈结构,增大胎冠帘线角度,调整胎面胶结构及配方设计等措施,有效提高了轮胎安全性能、耐磨性能和抗刺扎性能.优化设计后的轮胎充气外缘尺寸和胎面胶各项物理性能符合国家标准要求,经实际使用证明达到了设计要求,同时降低了成本,提高了市场竞争力.     

Computational design of thermoset nanocomposite coatings: Methodological study on coating development and testing

Thermoset nanocomposites (TSNCs) may offer significantly improved performance over conventional thermoset materials, and thus are attractive for wide industrial applications, especially in the coating industry. Design of TSNCs via experiment, however, faces various technical challenges due to design complexity. Computational design can provide deep insights and identify superior design solutions through exploring opportunities in a usually huge design space. This paper introduces a generic computational methodology for the design, characterization, and testing of TSNC-based coatings. A distinct feature of the methodology is its capability of generating quantitative correlations among material formulation, processing condition, coating microstructure and property, coating performance, and processing efficiency. The correlations can enable a comprehensive analysis for optimal TSNC coating design. Case studies will demonstrate the methodological efficacy and attractiveness.     

Initial Molecular Recognition Steps of McjA Precursor during Microcin J25 Lasso Peptide Maturation

Microcin J25 (MccJ25) has emerged as an excellent model to understand the maturation of ribosomal precursor peptides into the entangled lasso fold. MccJ25 biosynthesis relies on the post‐translational modification of the precursor McjA by the ATP‐dependent protease McjB and the lactam synthetase McjC. Here, using NMR spectroscopy, we showed that McjA is an intrinsically disordered protein without detectable conformational preference, which emphasizes the active role of the maturation machinery on the three‐dimensional folding of MccJ25. We further showed that the N‐terminal region of the leader peptide is involved in interaction with both maturation enzymes and identified a predominant interaction of V43–S55 in the core McjA sequence with McjC. Moreover, we demonstrated that residues K23–Q34 in the N‐terminal McjA leader peptide tend to adopt a helical conformation in the presence of membrane mimics, implying a role in directing McjA to the membrane in the vicinity of the lasso synthetase/export machinery. These data provide valuable insights into the initial molecular recognition steps in the MccJ25 maturation process.     

A method for computational combinatorial peptide design of inhibitors of Ras protein

A computational combinatorial approach is proposed for the designof a peptide inhibitor of Ras protein. The procedure involvesthree steps. First, a `Multiple Copy Simultaneous Search' identifiesthe location of specific functional groups on the Ras surface.This search method allowed us to identify an important bindingsurface consisting of two ß strands (residues 5–8and 52–56), in addition to the well known Ras effectorloop and switch II region. The two ß strands had not previouslybeen reported to be involved in Ras–Raf interaction. Second,after constructing the peptide inhibitor chain based on thelocation of N-methylacetamide (NMA) minima, functional groupsare selected and connected to the main chain C atom. This stepgenerates a number of possible peptides with different sequenceson the Ras surface. Third, potential inhibitors are designedbased on a sequence alignment of the peptides generated in thesecond step. This computational approach reproduces the conservedpattern of hydrophobic, hydrophilic and charged amino acidsidentified from the Ras effectors. The advantages and limitationsof this approach are discussed.