Structure and Kinetic Investigation of Streptococcus pyogenes Family GH38 α-Mannosidase

BACKGROUND: The enzymatic hydrolysis of alpha-mannosides is catalyzed by glycoside hydrolases (GH), termed alpha-mannosidases. These enzymes are found in different GH sequence-based families. Considerable research has probed the role of higher eukaryotic "GH38" alpha-mannosides that play a key role in the modification and diversification of hybrid N-glycans; processes with strong cellular links to cancer and autoimmune disease. The most extensively studied of these enzymes is the Drosophila GH38 alpha-mannosidase II, which has been shown to be a retaining alpha-mannosidase that targets both alpha-1,3 and alpha-1,6 mannosyl linkages, an activity that enables the enzyme to process GlcNAc(Man)(5)(GlcNAc)(2) hybrid N-glycans to GlcNAc(Man)(3)(GlcNAc)(2). Far less well understood is the observation that many bacterial species, predominantly but not exclusively pathogens and symbionts, also possess putative GH38 alpha-mannosidases whose activity and specificity is unknown. METHODOLOGY/PRINCIPAL FINDINGS: Here we show that the Streptococcus pyogenes (M1 GAS SF370) GH38 enzyme (Spy1604; hereafter SpGH38) is an alpha-mannosidase with specificity for alpha-1,3 mannosidic linkages. The 3D X-ray structure of SpGH38, obtained in native form at 1.9 A resolution and in complex with the inhibitor swainsonine (K(i) 18 microM) at 2.6 A, reveals a canonical GH38 five-domain structure in which the catalytic "-1" subsite shows high similarity with the Drosophila enzyme, including the catalytic Zn(2+) ion. In contrast, the "leaving group" subsites of SpGH38 display considerable differences to the higher eukaryotic GH38s; features that contribute to their apparent specificity. CONCLUSIONS/SIGNIFICANCE: Although the in vivo function of this streptococcal GH38 alpha-mannosidase remains unknown, it is shown to be an alpha-mannosidase active on N-glycans. SpGH38 lies on an operon that also contains the GH84 hexosaminidase (Spy1600) and an additional putative glycosidase. The activity of SpGH38, together with its genomic context, strongly hints at a function in the degradation of host N- or possibly O-glycans. The absence of any classical signal peptide further suggests that SpGH38 may be intracellular, perhaps functioning in the subsequent degradation of extracellular host glycans following their initial digestion by secreted glycosidases

An improved experimental test set-up to study the performance of granular columns

This paper describes an innovative design of a newly developed large test setup for testing the performance of footings supported on soft clay reinforced with granular columns. This advanced testing method is used to examine the settlement of footings supported on granular columns. Two important features of the equipment are (a) the axial loading system which allows samples to be consolidated under Ko condition while the load is applied onto a small foundation area of the sample, and (b) a relatively large sample size of 300-mm diameter and 400-mm high. The system is also equipped with pressure cells located beneath the footing and top cap to measure the pressure distribution with respect to foundation displacement and a lateral strain gage to monitor boundary effects. This paper reports on some of the early findings from the preliminary tests carried out using this equipment. Samples for testing were prepared by consolidating kaolin slurry in a large one-dimensional consolidation chamber. The granular columns were installed using the replacement method by compacting crushed basalt (uniformly graded with 90 % between 1.5–2-mm particle sizes) into a preformed hole. The preliminary tests have yielded promising results, validating the functionality of the equipment and support the prospect of increasing the knowledge with respect to settlement response and design of a footing supported on granular columns

Centrifuge Modelling With Transparent Soil and Laser Aided Imaging

Transparent synthetic soils have been developed as a soil surrogate to enable internal visualization of geotechnical processes in physical models. While significant developments have been made to enhance the methodology and capabilities of transparent soil modelling, the technique is not yet exploited to its fullest potential. Tests are typically conducted at 1 g in small bench size models, which invokes concerns about the impact of scale and stress level observed in previously reported work. This paper recognized this limitation and outlines the development of improved testing methodology whereby the transparent soil and laser aided imaging technique are translated to the centrifuge environment. This has a considerable benefit such that increased stresses are provided, which better reflect the prototype condition. The paper describes the technical challenges associated with implementing this revised experimental methodology, summarizes the test equipment/systems developed, and presents initial experimental results to validate and confirm the successful implementation and scaling of transparent soil testing to the high gravity centrifuge test environment. A 0.6 m wide prototype strip foundation was tested at two scales using the principle of “modelling of models,” in which similar performance was observed. The scientific developments discussed have the potential to provide a step change in transparent soil modelling methodology, crucially providing more representative stress conditions that reflect prototype conditions, while making a broader positive contribution to physical modelling capabilities to assess complex soil–structure boundary problems

Quantification of Optical Clarity of Transparent Soil Using the Modulation Transfer Function

Transparent synthetic soils have been developed as a soil surrogate to enable internal visualization of geotechnical processes in physical models. Transparency of the soil dictates the overarching success of the technique; however, despite this fundamental requirement, no quantitative framework has yet been established to appraise the visual quality of transparent soil. Previous approaches to assess and optimize transparency quality included an eye chart assessment method, although this approach is highly subjective and operator-dependent. In this paper, an independent method for quantitatively assessing the optical quality of transparent soil is proposed based on the optical calibration method, Modulation Transfer Function (MTF). The work explores this hypothesis and assesses the potential for MTF to quantify the optical quality of transparent soils for a number of aspects including (i) optimum oil blend ratio, (ii) depth of viewing plane, and (iii) temperature. The results confirmed that MTF offers a robust and reliable method to provide an independent quantitative measure of the optical quality of transparent soil. The impact of reduced soil transparency and the ability to track speckle patterns—thus accuracy and precision of displacement measurement—was correlated with MTF to evaluate the permissible viewing depth of transparent soil

Transparent soil to model thermal processes: An energy pile example

Managing energy resources is fast becoming a crucial issue of the 21st century, with groundbased heat exchange energy structures targeted as a viable means of reducing carbon emissions associated with regulating building temperatures. Limited information exists about the thermo-dynamic interactions of geothermal structures and soil owing to the practical constraints of placing measurement sensors in proximity to foundations; hence, questions remain about their long-term performance and interaction mechanics. An alternative experimental method using transparent soil and digital image analysis was proposed to visualize heat flow in soil. Advocating the loss of optical clarity as a beneficial attribute of transparent soil, this paper explored the hypothesis that temperature change will alter its refractive index and therefore progressively reduce its transparency, becoming more opaque. The development of the experimental methodology was discussed and a relationship between pixel intensity and soil temperature was defined and verified. This relationship was applied to an energy pile example to demonstrate heat flow in soil. The heating zone of influence was observed to extend to a radial distance of 1.5 pile diameters and was differentiated by a visual thermal gradient propagating from the pile. The successful implementation of this technique provided a new paradigm for transparent soil to potentially contribute to the understanding of thermo-dynamic processes in soil

The Development and Testing of an Instrumented Open-Ended Model Pile

This paper describes the development of a model instrumented open-ended (pipe) pile. The importance of model geometry and separating the shaft, annular and plug load, and horizontal effective stresses is discussed. A detailed description of the construction of the twin-walled open-ended pile is presented. Particular attention was given to protecting the fragile instrumentation from the rigours of installation and the effects of water ingress. Calibration procedures, which were used to verify the instrument reliability, are also discussed. The final section describes field tests conducted in both loose sand and medium-dense sand deposits, which are used to validate the instrument performance.Deposited by bulk importTS 22.02.1