A 96-well microtiter plate assay for high-throughput screening of Mycobacterium tuberculosis dTDP-d-glucose 4,6-dehydratase inhibitors

Mycobacterium tuberculosis dTDP-d-glucose 4,6-dehydratase (RmlB) is the second enzyme for the biosynthesis of dTDP-l-rhamnose, which is a sugar donor to the synthesis of the cell wall linker, d-N-acetylglucosamine-l-rhamnose. RmlB is essential to mycobacterial growth and is not found in humans; therefore, it is a potential target for developing new anti-tuberculosis drugs. So far, there has been no suitable method for high-throughput screening of RmlB inhibitors. Here, the recombinant M. tuberculosis RmlB was purified and an absorbance-based microtiter plate assay was developed for RmlB activity. It could be used for high-throughput screening of RmlB inhibitors. The kinetic properties of M. tuberculosis RmlB, including optimal pH, optimal temperature, the effect of metal ions, and the kinetic parameters, were determined with this assay. The inhibitory effects of dTTP and dTDP on M. tuberculosis RmlB were also studied with the assay.     

Enzymatic cycling method using creatine kinase to measure creatine by real-time detection

We have developed a novel enzymatic cycling method that uses creatine kinase (CK) to measure creatine. The method takes advantage of the reversibility of the CK reaction in which the forward (creatine phosphate forming) and reverse reactions are catalyzed in the presence of an excess amount of ATP and IDP, respectively. Real-time detection was accomplished using ADP-dependent glucokinase (ADP–GK) together with glucose-6-phosphate dehydrogenase. ADP, one of the cycling reaction products, was distinguished from IDP by using the nucleotide selectivity of the ADP–GK. The increasing level of ADP was measured from the level of reduced NADP at 340 nm. The method is appropriate for an assay that requires high sensitivity because the rate of increase in absorbance at 340 nm is proportional to the amount of CK present in the reaction mix. We reasoned that the method with CK in combination with creatinine amidohydrolase could be used to assay creatinine, an important marker of kidney function. Our results confirmed the quantitative capability of the assay.     

Ce3+/Tb3+ non‐/single‐/co‐doped K‐Lu‐F materials: synthesis,optical properties,and energy transfer

Using a hydrothermal method, Ce³⁺/Tb³⁺ non‐/single‐/co‐doped K‐Lu‐F materials have been synthesized. The X‐ray diffraction (XRD) results suggest that the Ce³⁺ and/or Tb³⁺ doping had great effects on the crystalline phases of the final samples. The field emission scanning electron microscopy (FE‐SEM) images indicated that the samples were in hexagonal disk or polyhedron morphologies in addition to some nanoparticles, which also indicated that the doping also had great effects on the sizes and the morphologies of the samples. The energy‐dispersive spectroscopy (EDS) patterns illustrated the constituents of different samples. The enhanced emissions of Tb³⁺ were observed in the Ce³⁺/Tb³⁺ co‐doped K‐Lu‐F materials. The energy transfer (ET) efficiency η_T were calculated based on the fluorescence yield. The ET mechanism from Ce³⁺ to Tb³⁺ was confirmed to be the dipole–quadrupole interaction inferred from the theoretical analysis and the experimental data. Copyright © 2015 John Wiley & Sons, Ltd.     

Assessing the importance of environmental factors to phytoliths of Phragmites communis in north-eastern China

Phytoliths have proved to be reliable indicators of environmental conditions both at present and in the past, and can provide evidence for the distribution of taxa or vegetation. Understanding the environmental significance of phytoliths within plants helps in drawing more reliable inferences about palaeovegetation and in reconstructing the palaeo-environment. The present study examined the relationship between phytoliths and environmental factors to assess the environmental significance of different types of phytoliths. Phytoliths were extracted from Phragmites communis growing in xerophytic and aquatic habitats at twelve sampling sites in north-eastern China and their implications for the environment were assessed using quantitative data mainly including phytolith concentration and environmental factors and statistical analyses. Principal component analysis (PCA) of several environmental factors (including the climate, micro terrain, and the physicochemical properties of soils) revealed that other factors being constant, P. communis phytolith concentrations were influenced largely by the average annual temperature and precipitation. Orthogonal experiment analysis and three-way analyses of variance of the concentrations confirmed the reliability of the results of the PCA. More specifically, the concentrations of the saddle and rondel types of phytoliths (the short-cell phytoliths) were closely linked to the average annual temperature whereas those of the elongate, lanceolate, and bulliform phytoliths (the non-short-cell phytoliths) were more sensitive to the variations in water status. The results contribute to further confirming the major environmental implications of phytoliths and, in turn, providing a reference for growth and development of wetland plants. Our findings also provide critical information that could help managers and policymakers assess and modify ecological restoration practices.     

Role of solvent properties of aqueous media in macromolecular crowding effects

Analysis of the macromolecular crowding effects in polymer solutions show that the excluded volume effect is not the only factor affecting the behavior of biomolecules in a crowded environment. The observed inconsistencies are commonly explained by the so-called soft interactions, such as electrostatic, hydrophobic, and van der Waals interactions, between the crowding agent and the protein, in addition to the hard nonspecific steric interactions. We suggest that the changes in the solvent properties of aqueous media induced by the crowding agents may be the root of these “soft” interactions. To check this hypothesis, the solvatochromic comparison method was used to determine the solvent dipolarity/polarizability, hydrogen-bond donor acidity, and hydrogen-bond acceptor basicity of aqueous solutions of different polymers (dextran, poly(ethylene glycol), Ficoll, Ucon, and polyvinylpyrrolidone) with the polymer concentration up to 40% typically used as crowding agents. Polymer-induced changes in these features were found to be polymer type and concentration specific, and, in case of polyethylene glycol (PEG), molecular mass specific. Similarly sized polymers PEG and Ucon producing different changes in the solvent properties of water in their solutions induced morphologically different α-synuclein aggregates. It is shown that the crowding effects of some polymers on protein refolding and stability reported in the literature can be quantitatively described in terms of the established solvent features of the media in these polymers solutions. These results indicate that the crowding agents do induce changes in solvent properties of aqueous media in crowded environment. Therefore, these changes should be taken into account for crowding effect analysis.     

Finite element modelling approaches for well-ordered porous metallic materials for orthopaedic applications: cost effectiveness and geometrical considerations

The mechanical properties of well-ordered porous materials are related to their geometrical parameters at the mesoscale. Finite element (FE) analysis is a powerful tool to design well-ordered porous materials by analysing the mechanical behaviour. However, FE models are often computationally expensive. This article aims to develop a cost-effective FE model to simulate well-ordered porous metallic materials for orthopaedic applications. Solid and beam FE modelling approaches are compared, using finite size and infinite media models considering cubic unit cell geometry. The model is then applied to compare two unit cell geometries: cubic and diamond. Models having finite size provide similar results than the infinite media model approach for large sample sizes. In addition, these finite size models also capture the influence of the boundary conditions on the mechanical response for small sample sizes. The beam FE modelling approach showed little computational cost and similar results to the solid FE modelling approach. Diamond unit cell geometry appeared to be more suitable for orthopaedic applications than the cubic unit cell geometry.     

鳖源胶原蛋白提取纯化及在生物材料中的应用

近年来,胶原蛋白因其良好的生物学性能在生物材料应用中得到越来越多的关注,为了建立一种快速高效的鳖源胶原蛋白纯化方法和探究其在生物材料中的应用价值,首先用Van Gieson染色法和苦味酸-天狼星红染色法观察裙边胶原纤维组织结构,发现裙边胶原纤维含量非常高且类型主要是Ⅰ型。采用不同截留分子量的透析袋对裙边胶原蛋白粗提液进行直接透析纯化,发现截留分子量为100 k Da的透析袋在透析48 h后对裙边胶原蛋白的纯化效果最好,SDS-PAGE检验显示几乎没有杂带。对裙边胶原蛋白生物学性能包括吸水性、体外降解性进行考察,其吸水力和持水力分别高达12.06 g/g和98.21%,且在72 h后被完全降解;对裙边胶原蛋白海绵的溶血性、皮肤致敏性、肝部创伤止血性、促创伤皮肤愈合性进行了研究,并和交联胶原进行比较,发现裙边胶原蛋白和交联胶原均不会造成SD大鼠溶血和皮肤过敏,二者均具有良好的止血效果,且裙边胶原蛋白能显著缩短创伤皮肤愈合时间,具有良好的促创伤皮肤愈合性,而交联胶原效果欠佳。本研究表明裙边胶原蛋白表现出了优良的生物学性能,在生物材料领域具有很大应用价值。     

Donor substrate promiscuity of bacterial β1–3-N-acetylglucosaminyltransferases and acceptor substrate flexibility of β1–4-galactosyltransferases

β1–3-N-Acetylglucosaminyltransferases (β3GlcNAcTs) and β1–4-galactosyltransferases (β4GalTs) have been broadly used in enzymatic synthesis of N-acetyllactosamine (LacNAc)-containing oligosaccharides and glycoconjugates including poly-LacNAc, and lacto-N-neotetraose (LNnT) found in the milk of human and other mammals. In order to explore oligosaccharides and derivatives that can be synthesized by the combination of β3GlcNAcTs and β4GalTs, donor substrate specificity studies of two bacterial β3GlcNAcTs from Helicobacter pylori (Hpβ3GlcNAcT) and Neisseria meningitidis (NmLgtA), respectively, using a library of 39 sugar nucleotides were carried out. The two β3GlcNAcTs have complementary donor substrate promiscuity and 13 different trisaccharides were produced. They were used to investigate the acceptor substrate specificities of three β4GalTs from Neisseria meningitidis (NmLgtB), Helicobacter pylori (Hpβ4GalT), and bovine (Bβ4GalT), respectively. Ten of the 13 trisaccharides were shown to be tolerable acceptors for at least one of these β4GalTs. The application of NmLgtA in one-pot multienzyme (OPME) synthesis of two trisaccharides including GalNAcβ1–3Galβ1–4GlcβProN₃ and Galβ1–3Galβ1–4Glc was demonstrated. The study provides important information for using these glycosyltransferases as powerful catalysts in enzymatic and chemoenzymatic syntheses of oligosaccharides and derivatives which can be useful probes and reagents.     

Ultrasonic Welding of Thermoplastic Composite Coupons for Mechanical Characterization of Welded Joints through Single Lap Shear Testing

This paper presents a novel straightforward method for ultrasonic welding of thermoplastic-composite coupons in optimum processing conditions. The ultrasonic welding process described in this paper is based on three main pillars. Firstly, flat energy directors are used for preferential heat generation at the joining interface during the welding process. A flat energy director is a neat thermoplastic resin film that is placed between the parts to be joined prior to the welding process and heats up preferentially owing to its lower compressive stiffness relative to the composite substrates. Consequently, flat energy directors provide a simple solution that does not require molding of resin protrusions on the surfaces of the composite substrates, as opposed to ultrasonic welding of unreinforced plastics. Secondly, the process data provided by the ultrasonic welder is used to rapidly define the optimum welding parameters for any thermoplastic composite material combination. Thirdly, displacement control is used in the welding process to ensure consistent quality of the welded joints. According to this method, thermoplastic-composite flat coupons are individually welded in a single lap configuration. Mechanical testing of the welded coupons allows determining the apparent lap shear strength of the joints, which is one of the properties most commonly used to quantify the strength of thermoplastic composite welded joints.